2009. "Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput." Journal of Chromatography B 877(8-9):663-670. Abstract We report on the development and characterization of an automated metal-free nanoscale multiple-capillary system for reversed-phase liquid chromatography-mass spectrometry analysis of phosphopeptides. The system incorporates a capillary column (50 um i.d. × 30 cm, packed with 5 um C18 particles) coupled on-line to a solid phase extraction column (150 um i.d. × 4 cm, packed with 5 um C18 particles). Electrospray ionization tips are constructed on the packed capillary column to couple the reversed-phase liquid chromatographic separation to a linear ion trap tandem mass spectrometer.
2009. "Combined Pulsed-Q dissociation and electron transfer dissociation for identification and quantitation of iTRAQ–labeled phosphopeptides." Analytical Chemistry 81(10):4137-4143. doi:10.1021/ac802605m Abstract Multiplex isobaric tags for relative and absolute quantification (iTRAQ) enable high-throughput quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. A challenging but highly promising application is to analyze iTRAQ-labeled peptides using a sensitive linear ion trap mass spectrometer (LTQ-MS) and pulsed Q dissociation (PQD), a form of ion trap collision activated dissociation (CAD) designed to allow detection of low mass-to-charge fragment ions. Electron dissociation transfer (ETD), on the other hand, is complementary to PQD and is especially useful for sequencing peptides containing post-translational modifications (PTMs). Here, we developed an integrated workflow for robust and accurate quantitative identification of iTRAQ labeled phosphopeptides that integrates the PQD and ETD fragmentation methods together with PQD optimization, data management and bioinformatics tools. Analysis of the phosphoproteome of human fibroblast cells demonstrated that this hybrid mode is superior to either PQD or ETD alone for phosphopeptide identification and quantitation. The combined PQD/ETD approach can qualitatively identify additional phosphopeptides than ETD alone and PQD information can provide better quantitation of ETD identified iTRAQ-labeled phosphopeptides.
2009. "An integrated workflow for characterizing intact phosphoproteins from complex mixtures." Analytical Chemistry 81(11):4210-4219. doi:10.1021/ac802487q Abstract The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the "state" of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography-mass spectrometry (LC-MS) analysis of intact phosphoproteins. The technology platform built upon integrated bottom-up and top-down approach that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection.
2009. "An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications." Journal of Proteome Research 8(3):1347-1357. Abstract We present an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs high resolution reversed phase (RP) LC separations coupled on-line with a 12T Fourier transform ion cyclotron resonance (FTICR) spectrometer to profile and tentatively identify modified proteins, using detected intact protein masses in conjunction with bare protein identifications from the bottom-up analysis of the same fraction. Selected identifications are incorporated into a target ion list for subsequent offline gas phase fragmentation that uses only an aliquot of the original fraction used for bottom-up analysis.
2009. "Regulation of the Low Dose Radiation Paracrine-Specific Anchorage-Independent Growth Response by Annexin A2." Radiation Research 172(1):96-105. doi:10.1667/RR1220.1 Abstract ABSTRACT-Here we identify release of annexin A2 into the culture medium in response to low dose X-ray radiation exposure and establish functional linkages to an established paracrine factor-mediated anchorage-independent growth response. Using a standard bicameral coculture model, we observe that annexin A2 levels associated with non-irradiated neighboring cells seeded in the lower chamber (annexin A2 silenced [shRNA] JB6 cells) are increased upon coculture with irradiated (10-50 cGy) JB6 cells seeded in the upper chamber, relative to coculture with sham exposed JB6 cells seeded in the upper chamber, suggesting that annexin A2 released into the medium is capable of communicating in a paracrine fashion. Using a previously established coculture model, we observed that the paracrine factor-mediated anchorage-independent growth response to low dose X-ray radiation is markedly reduced when irradiated annexin A2 silenced (shRNA) JB6 cells are used, relative to coculture with irradiated annexin A2 competent vector control counterparts. These observations suggest that annexin A2 is functionally linked to the radiation paracrine factor-specific anchorage-independent growth response in JB6 cells.
2009. "FT-ICR MS optimization for the analysis of intact proteins." International Journal of Mass Spectrometry 287(1-3 SP ISS):32-38. doi:10.1016/j.ijms.2008.10.010 Abstract Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) remains the technique of choice for the analysis of intact proteins from complex biological systems, i.e. top-down proteomics. Recently, we have implemented a compensated open cylindrical ion trapping cell into a 12 T FT-ICR mass spectrometer. This new cell has previously demonstrated improved sensitivity, dynamic range, and mass measurement accuracy for the analysis of relatively small tryptic peptides. These improvements are due to the improved trapping potential of the cell which is significantly closer to the ideal harmonic trapping potential. Here we report the instrument optimization for the analysis of large macro-molecular ions, such as proteins. Also, presented are first principle theoretical considerations to account for different optimum conditions for the analysis of large macro-molecules. The proposed high energy ion loss mechanism is further supported by experimental results of bovine ubiquitin and serum albumin. We find that the analysis of large macro-molecules can be significantly improved by the further reduction of pressure in the ion trapping cell. This will reduce the impact of the high energy ion loss mechanism and enable increased sensitivity and mass measurement accuracy to be realized without compromising resolution. Further, these results appear to be applicable to FTMS in general, and the high energy ion loss mechanism applies to Orbitrap mass analyzers as well.
2009. "Coulombic Effects on Ion Mobility Spectrometry ." Analytical Chemistry 81(12):4778-4787. Abstract The ion mobility spectrometry (IMS) is now taking its place among widely applied analytical methods. When coupled with mass spectrometers (MS), IMS becomes a powerful analytical tool for separating complex samples and investigating molecular structure, and improvements of IMS-MS instrumentation, e.g. to IMS resolving power and sensitivity, are highly desirable. Implementation of an ion trap for accumulation and pulsed ion injection to IMS based on the ion funnel has provided considerably increased ion currents, and thus a basis for improved sensitivity and (indirectly) measurement throughput. However, large ion populations may manifest Coulombic effects contributing to the spatial dispersion of ions traveling in the IMS drift tube, and thus affect IMS resolving power. In this study we present an analysis of Coulombic effects on IMS resolution. Basic relationships have been obtained for the spatial evolution of ion packets due to Coulombic repulsion. The theoretical relationships were compared with results of a computer model that simulates IMS operation based on a first principles approach. Initial experimental results reported here are consistent with the computer modeling and these relationships. A noticeable decrease of the IMS resolving power was observed for specific ion populations of >10,000 elementary charges. IMS operation conditions to minimize Coulombic effects, while minimizing sacrifices to performance, are discussed.
2009. " Transport Functions Dominate the SAR11 Metaproteome at Low-Nutrient Extremes in the Sargasso Sea." The ISME Journal 3(1):93-105. Abstract The northwestern Sargasso Sea is part of the North Atlantic subtropical oceanic gyre that is characterized as seasonally oligotrophic with pronounced stratification in the summer and autumn. Essentially a marine desert, the biological productivity of this region is reduced during stratified periods as a result of low concentrations of phosphorous and nitrogen in the euphotic zone. To better understand the mechanisms of microbial survival in this oligotrophic environment, we used capillary LC-tandem mass spectrometry to study the composition of microbial proteomes in surface samples collected in September 2005. A total of 2279 peptides that mapped to 236 SAR11 proteins, and 3208 peptides that mapped to 404 Synechococcus proteins, were detected. Mass spectra from SAR11 periplasmic binding proteins accounted for a disproportionately large fraction of the peptides detected, consistent with observations that these extremely small cells devote a large proportion of their volume to periplasm. Abundances were highest for periplasmic substrate-binding proteins for phosphate, amino acids, phosphonate, sugars, and spermidine. Although the data showed that a large fraction of microbial protein synthesis in the Sargasso Sea is devoted to inorganic and organic nutrient acquisition, the proteomes of both SAR11 and Synechococcus also indicated that these populations were actively growing. Our findings support the view that competition for multiple nutrients in oligotrophic systems is extreme but sufficient to sustain microbial community activity.
2009. "Ultrafast Differential Ion Mobility Spectrometry at Extreme Electric Fields in Multichannel Microchips ." Analytical Chemistry 81(15):6489-6495. doi:10.1021/ac900892u Abstract The maximum electric field intensity (E) in FAIMS analyses was doubled to E ~60 kV/cm. Such strong fields cause electrical breakdown in ~1 mm gaps (for nearly all gases) and thus were impossible in earlier devices with 0.5 - 2.5 mm gaps. However, thinner gaps permit higher E, and >60 kV/cm was established here in semiconductor microchips with 35-μm gaps. As FAIMS efficiency is exceptionally sensitive to E, such extreme values allow accelerating analyses at same resolution by orders of magnitude. Here we demonstrate FAIMS ion filtering in ~20 μs, or ~1% of the previously needed time. The resolving power, though short of that for “macroscopic” FAIMS units, suffices for many applications. Microscopic gaps also enable concurrent ion processing in multiple (here, 47) channels, which greatly relaxes the charge capacity constraints of planar FAIMS approach. These microchips were integrated into stand-alone FAIMS and FAIMS/MS instruments with ions supplied by β-radiation or electrospray sources, and evaluated in separations of small molecules, amino acids, and peptides. Present FAIMS analyzer can employ various pure and mixed gases, though the results depend on gas composition less than those with FAIMS using lower E. The measured separation performance is broadly in agreement with the first-principles modeling of FAIMS operation
2009. " Pendular Proteins in Gases and New Avenues for Characterization of Macromolecules by Ion Mobility Spectrometry ." Proceedings of the National Academy of Sciences of the United States of America 106(16):6495-6500. doi:doi: 10.1073/pnas.0812318106 Abstract Polar molecules align in electric fields when the dipole energy (proportional to field intensity E × dipole moment p) exceeds the thermal rotational energy. Small molecules have low p and align only at inordinately high E or upon extreme cooling. Many biomacromolecules and ions are strong permanent dipoles and may align at E achievable in gases and room temperature. The collision cross sections of aligned ions with gas molecules generally differ from orientationally averaged quantities, affecting ion mobilities measured in ion mobility spectrometry (IMS). Field asymmetric waveform IMS (FAIMS) separates ions by the difference between mobilities at high and low E and hence can resolve and identify macroion conformers based on the mobility difference between pendular and free rotor states. An exceptional sensitivity of that difference to the ion geometry and charge distribution holds the potential for a powerful new method for separation and characterization of macromolecular species. Theory predicts that the pendular alignment of ions in gases at any E requires a minimum p depending on the ion mobility, gas pressure, and temperature. At ambient conditions used in current FAIMS systems, the p for realistic ions must exceed ~300 - 400 Debye. The dipole moments of proteins statistically increase with increasing mass, and such values are typical above ~30 kDa. FAIMS analyses of protein ions and complexes of ~30 - 130 kDa show an order-of-magnitude expansion of separation space compared to smaller proteins and other ions, consistent with expectations for the dipole-aligned regime.
2009. "Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica." Infection and Immunity 77(8):3227-3233. doi:10.1128/IAI.00063-09 Abstract Macrophages plan important roles in controlling Salmonella-mediated systemic infection. To investigate the responses of macrophages to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serovar Typhimurium (STM) and then performed a comparative liquid chromatography-tandem mass spectrometry [LC-MS(/MS)]-based proteomics analysis of the infected macrophages. A total of 1006 macrophage and 115 STM proteins were indentified from this study. Most of STM proteins were found at late stage of the time course of infection, consistent with the fact that STM proliferates inside RAW 264.7 macrophages. Majority of the identified macrophage proteins were house keeping-related, including cytoplasmic superoxide dismutase 1 (SOD1), whose peptide abundances were relatively constant during the time course of infection. Compared to those in no infection control, the peptide abundances of 244 macrophage proteins (or 24% of total indentified macrophage proteins) changed considerably after STM infection. The functions of these STM infection-affected macrophage proteins were diverse and ranged from production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase or iNOS) or production of prostaglandin H2 (i.e., prostaglandin-endoperoxide synthase 2, also know as cyclooxygenase-2 or COX-2) to regulation of intracellular traffic (e.g., sorting nexin or SNX 5, 6 and 9), demonstrating a global impact of STM infection on macrophage proteome. Western-blot analysis not only confirmed the LC-MS(/MS) results of SOD1, COX-2 and iNOS, but also revealed that the protein abundances of mitochondrial SOD2 increased after STM infection, indicating an infection-induced oxidative stress in mitochondria.
2009. "Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample ." Journal of Proteome Research 8(1):290-299. Abstract Quantitative comparison of protein abundances across a relatively large number of patient samples is an important challenge for clinical proteomic applications. Herein we describe a dual-quantitation strategy that allows the simultaneous integration of complementary label-free and stable isotope labeling based approaches without increasing the number of LC-MS analyses. The approach utilizes a stable isotope 18O-labeled “universal” reference sample as a comprehensive set of internal standards spiked into each individually processed unlabeled patient sample. The quantitative data are based on both the direct 16O-MS intensities for label-free quantitation and the 16O/18O isotopic peptide pair ratios that compare each patient sample to the identical labeled reference. The effectiveness of this dual-quantitation approach for large scale quantitative proteomics is demonstrated by the application to a set of 38 clinical plasma samples from surviving and non-surviving severe burn patients. With the coupling of immunoaffinity depletion, cysteinyl-peptide enrichment based fractionation, high resolution LC-MS measurements, and the dual-quantitation approach, a total of 318 proteins were confidently quantified with at least two peptides and 263 proteins were quantified by both approaches. The strategy also enabled a direct comparison between the two approaches with the labeling approach showing significantly better precision in quantitation while the label-free approach resulted in more protein identifications. The relative abundance differences determined by the two approaches also show strong correlation. Finally, the dual-quantitation strategy allowed us to identify more candidate protein biomarkers, illustrating the complementary nature of the two quantitative methods.
2009. "Selection of the Optimum Electrospray Voltage for Gradient Elution LC-MS Measurements." Journal of the American Society for Mass Spectrometry 20(4):682-688. Abstract Changes in liquid composition during gradient elution liquid chromatography (LC) and mass spectrometry (MS) analyses affect the electrospray operation. To establish methodologies for judicious selection of the electrospray voltage, we monitored in real-time the effect of the LC gradient on the spray current. The optimum range of the electrospray voltage shifted to lower values as the concentration of organic solvent in the eluent increased during reversed-phase LC analyses. These results provided the means to rationally select the voltage that ensured successful electrospray operation throughout gradient elution LC-MS experiments. A small run-to-run drift in the spray current was observed for electrosprays operated at constant voltage. This could be the result of fouling or degradation of the electrospray emitter, which affected the electric field driving the electrospray. Algorithms using feedback from spray current measurements to maintain the electrospray voltage within the optimum operating range throughout gradient elution LC-MS were evaluated. The electrospray operation with voltage regulation and at constant, judiciously selected voltage during gradient elution LC-MS measurements produced data with similar reproducibility.
2009. "Improving FAIMS Sensitivity using a Planar Geometry with Slit Interfaces." Journal of the American Society for Mass Spectrometry 20(9):1768-1774. doi:10.1016/j.jasms.2009.05.019 Abstract Differential mobility spectrometry or field asymmetric waveform ion mobility spectrometry (FAIMS) is gaining broad acceptance for analyses of gas-phase ions, especially in conjunction with largely orthogonal separation methods such as mass spectrometry (MS) and/or conventional (drift tube) ion mobility spectrometry. In FAIMS, ions are filtered while passing through a gap between two electrodes that may have planar or curved (in particular, cylindrical) geometry. Despite substantial inherent advantages of the planar configuration and its universal acceptance in stand-alone FAIMS devices, commercial FAIMS/MS systems have employed curved FAIMS geometries that could be interfaced to MS more effectively. Here we report a new planar (p-) FAIMS design with slit-shaped entrance and exit apertures that substantially increase ion transmission in and out of the analyzer. The front slit interface effectively couples p-FAIMS to multi-emitter electrospray ionization (ESI) sources, improving greatly the ion current introduced to the device. The back slit interface increases the transmission of ribbon-shaped ion beams output by the p-FAIMS to downstream stages such as a MS. Overall, the ion signal in ESI/FAIMS/MS analyses is raised by over an order of magnitude without affecting the FAIMS resolution.
2009. "Evaluation of a High Intensity Focused Ultrasound-Immobilized Trypsin Digestion and 18 O-Labeling Method for Quantitative Proteomics." Analytical Chemistry 81(15):6272-6277. Abstract A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in <1 min and minimized the amount of enzyme required compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from bacteria Shewanella oneidensis, and mouse plasma, as well as for the labeling of complex protein mixtures, which validated the application of this method for differential proteomic measurements. This approach is simple, reproducible, cost effective, and rapid, and thus well-suited for automation.
2009. "Highly Stable Trypsin-Aggregate Coatings on Polymer Nanofibers for Repeated Protein Digestion." Proteomics 9(7):1893-1900. Abstract A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This new process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was also resistant to autolysis, enabling repeated digestions of bovine serum albumin over 40 days and successful peptide identification by LC-MS/MS. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e. chymotrypsin), which makes it suitable for use in “real-world” proteomic applications. Overall, the biocatalytic nanofibers with enzyme aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.
2009. "Dilution-Free Analysis from Picoliter Droplets by Nano-Electrospray Ionization Mass Spectrometry." Angewandte Chemie International Edition 48(37):6832-6835. doi:10.1002/anie.200902501 Abstract The expanding role of microfluidics for chemical and biochemical analysis is due to factors including the favorable scaling of separation performance with reduced channel dimensions,[1] flexibility afforded by computer-aided device design, and the ability to integrate multiple sample handling and analysis steps into a single platform.[2] Such devices enable smaller liquid volumes and sample sizes to be handled than can be achieved on the benchtop, where sub-microliter volumes are difficult to work with and where sample losses to the surfaces of multiple reaction vessels become prohibitive. A particularly attractive microfluidic platform for sample-limited analyses employs aqueous droplets or plugs encapsulated by an immiscible oil.[3,4] Each droplet serves as a discrete compartment or reaction chamber enabling, e.g., high throughput screening[5,6] and kinetic studies[7-9] of femto- to nanoliter samples, as well as the encapsulation[10-12] and lysis[10] of individual cells with limited dilution of the cellular contents
2009. "AA statistical framework for protein quantitation in bottom-up MS-based proteomics ." Bioinformatics 25(16):2028-2034. doi:10.1093/bioinformatics/btp362 Abstract ABSTRACT Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and confidence measures. Challenges include the presence of low-quality or incorrectly identified peptides and widespread, informative, missing data. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model for protein abundance in terms of peptide peak intensities, applicable to both label-based and label-free quantitation experiments. The model allows for both random and censoring missingness mechanisms and provides naturally for protein-level estimates and confidence measures. The model is also used to derive automated filtering and imputation routines. Three LC-MS datasets are used to illustrate the methods. Availability: The software has been made available in the open-source proteomics platform DAnTE (Polpitiya et al. (2008)) (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu
2009. "Decon2LS: An Open-Source Software Package for Automated Processing and Visualization of High Resolution Mass Spectrometry Data." BMC Bioinformatics 10:, Abstract Data generated from liquid chromatography coupled to high-resolution mass spectrometry (LC-MS)-based studies of a biological sample can contain large amounts of biologically significant information in the form of proteins, peptides, and metabolites. Interpreting these data uses the mass spectra to infer masses and abundances of biomolecules injected into the instrument. Because of the inherent complexity of mass spectral patterns produced by these biomolecules, the analysis is significantly enhanced by using visualization capabilities to inspect and confirm results. In this paper we describe Decon2LS, an open-source software package for automated processing and visualization of high-resolution MS data. Drawing extensively on algorithms developed over the last ten years for ICR2LS, Decon2LS packages the algorithms as a rich set of modular, reusable processing classes for performing diverse functions such as raw data readers, routine peak finding, theoretical isotope distribution modelling, and deisotoping. Because the source code is openly available, these functionalities can now be used to build derivative applications in relatively fast manner. In addition, Decon2LS provides an extensive set of visualization tools, such as high performance chart controls.
2009. "An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells." Journal of Proteome Research 8(8):3852-3861. Abstract Protein tyrosine phosphorylation is a central regulatory mechanism in cell signaling. To extensively characterize the site-specific tyrosine phosphorylation in human cells, we present here a global survey of tyrosine phosphorylation sites in a normal-derived human mammary epithelial cell (HMEC) line by applying anti-phosphotyrosine (pTyr) peptide immunoaffinity purification (IP) coupled with high sensitivity LC-MS/MS. A total of 481 tyrosine phosphorylation sites (covered by 716 unique peptides) from 285 proteins were confidently identified in HMEC following the analysis of both the basal condition and an acute stimulated condition with epidermal growth factor (EGF). The estimated false discovery rate is 1.0% as measured by comparison against a scrambled database search. Comparison of these data to the literature showed significant agreement in site matches. Additionally 281 sites were not previously observed in HMEC culture were found. Twenty-nine of these sites have not been reported in any human cell or tissue system. The global profiling also allowed us to examine the phosphorylation stoichiometry differences based on spectral count information. Comparison of the data to a previous global proteome profiling study illustrates that most of the highly phoshorylated proteins are of relatively low-abundance. Large differences in phosphorylation stoichiometry for sites within the same protein were also observed for many of the identified proteins, suggesting potentially more important functional roles for those highly phosphorylated pTyr sites within a given protein. By mapping to major signaling networks such as EGF receptor and insulin growth factor-1 receptor signaling pathways, many known proteins involved in these pathways were revealed to be tyrosine phosphorylated, which should allow us to select interesting targeted involved in a given pathway for more directed studies. This extensive HMEC tyrosine phosphorylation dataset represents an important database resource for facilitating future hypothesis-driven or targeted quantitative studies involving tyrosine phosphorylation in HMEC or other human systems.
2009. "A method for investigating protein-protein interactions related to Salmonella typhimurium pathogenesis ." Journal of Proteome Research 8(3):1504-1514 . Abstract We successfully modified an existing method to investigate protein-protein interactions in the pathogenic bacterium Salmonella typhimurium (STM). This method includes i) addition of a histidine-biotin-histidine tag to the bait proteins via recombinant DNA techniques; ii) in vivo cross-linking with formaldehyde; iii) tandem affinity purification of bait proteins under fully denaturing conditions; and iv) identification of the proteins cross-linked to the bait proteins by liquid-chromatography in conjunction with tandem mass-spectrometry. In vivo cross-linking stabilized protein interactions permitted the subsequent two-step purification step conducted under denaturing conditions. The two-step purification greatly reduced nonspecific binding of non-cross-linked proteins to bait proteins. Two different negative controls were employed to reduce false-positive identification. In an initial demonstration of this approach, we tagged three selected STM proteins― HimD, PduB and PhoP― with known binding partners that ranged from stable (e.g., HimD) to transient (i.e., PhoP). Distinct sets of interacting proteins were identified with each bait protein, including the known binding partners such as HimA for HimD, as well as anticipated and unexpected binding partners. Our results suggest that novel protein-protein interactions may be critical to pathogenesis by Salmonella typhimurium. .
2009. "Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation." PLoS One 4(3):e4809. doi:10.1371/journal.pone.0004809 Abstract In recent years the profound importance of sRNA-mediated translational/post-transcriptional regulation has been increasingly appreciated. However, the global role played by translational regulation in control of gene expression has never been elucidated in any organism for the simple reason that global proteomics methods required to accurately characterize post-transcriptional processes and the knowledge of translational control mechanisms have only become available within the last few years. The proteins Hfq and SmpB are essential for the biological activity of a range of regulatory sRNAs and thus provide a means to identify potential targets of sRNA regulation. We performed a sample-matched global proteomics and transcriptional analysis to examine the role of Hfq and SmpB in global protein translation and virulence using the Salmonella typhimurium model system. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all Salmonella proteins, respectively, with limited correlation between transcription and protein expression. This is the first report suggesting that SmpB could be a general translational regulator. The broad spectrum of proteins modulated by Hfq was also surprising including central metabolism, LPS biosynthesis, two-component regulatory systems, quorum sensing, SP1-TTSS, oxidative stress, fatty acid metabolism, nucleoside and nucleotide metabolism, envelope stress, aminoacyl-tRNA synthetases, amino acid biosynthesis, peptide transport, and motility.. The extent of global regulation of translation by Hfq is unexpected, with profound effects in all stages of Salmonella’s life cycle. Our results represent the first global systems-level analysis of translational regulation; the elucidated potential targets of sRNA regulation from our analysis will facilitate the characterization of sRNA control mechanisms and their targets mRNAs by the broader scientific community by providing global-scale insights.
2009. "Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets ." Diabetes 58(11):2607-2615. Abstract The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.
2008. "Two-Dimensional Ion Mobility Analyses of Proteins and Peptides." Chapter 26 in Mass Spectrometry of Proteins and Peptides: Methods and Protocols, Methods in Molecular Biology, vol. 492, ed. MS Lipton and L Pasa-Tolic, pp. 417-445. Humana Press, Totowa, NJ. Abstract INTRODUCTION-Analytical methods based on ion transport in gases under the influence of electric field, known collectively as Ion Mobility Spectrometry (IMS), add major new capabilities to mass-spectrometric (MS) investigations. Separation of ion mixtures and characterization of ions by IMS are of particular utility in biological MS, where the sample complexity creates a virtually open-ended demand for separation power (1) while the ion size normally rules out structural elucidation by spectroscopic means. The use of IMS in mass spectrometry of proteins and peptides has expanded dramatically since the previous edition of this volume in 2000...
2008. "Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane." Journal of Proteome Research 7(5):2025-2032. doi:10.1021/pr700763r Abstract Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.
2008. "Improved Methods for the Enrichment and Analysis of Glycated Peptides ." Analytical Chemistry 80(24):9822-9829. doi:10.1021/ac801704j Abstract Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. Herein we report improved methods for the enrichment and analysis of glycated peptides using boronate affinity chromatography and electron transfer dissociation mass spectrometry, respectively. The enrichment of glycated peptides was improved by replacing an off-line desalting step with an on-line wash of column-bound glycated peptides using 50 mM ammonium acetate. The analysis of glycated peptides by MS/MS was improved by considering only higher charged (3) precursor-ions during data-dependent acquisition, which increased the number of glycated peptide identifications. Similarly, the use of supplemental collisional activation after electron transfer (ETcaD) resulted in more glycated peptide identifications when the MS survey scan was acquired with enhanced resolution. In general, acquiring ETD-MS/MS data at a normal MS survey scan rate, in conjunction with the rejection of both 1+ and 2+ precursor-ions, increased the number of identified glycated peptides relative to ETcaD or the enhanced MS survey scan rate. Finally, an evaluation of trypsin, Arg-C, and Lys-C showed that tryptic digestion of glycated proteins was comparable to digestion with Lys-C and that both were better than Arg-C in terms of the number glycated peptides identified by LC-MS/MS.
2008. "Analysis of Non-Enzymatically Glycated Peptides: Neutral-Loss Triggered MS3 Versus Multi-Stage Activation Tandem Mass Spectrometry." Rapid Communications in Mass Spectrometry 22(19):3027-3034. doi:10.1002/rcm.3703 Abstract Non-enzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. While electron transfer dissociation (ETD) has been shown to outperform collision-induced dissociation (CID) in sequencing glycated peptides by tandem mass spectrometry, ETD instrumentation is not yet available in all laboratories. In this study, we evaluated different advanced CID techniques (i.e., neutral-loss triggered MS3 and multi-stage activation) during LC-MSn analyses of Amadori-modified peptides enriched from human serum glycated in vitro. During neutral-loss triggered MS3 experiments, MS3 scans triggered by neutral-losses of 3 H2O or 3 H2O + HCHO produced similar results in terms of glycated peptide identifications. However, neutral losses of 3 H2O resulted in significantly more glycated peptide identifications during multi-stage activation experiments. Overall, the multi-stage activation approach produced more glycated peptide identifications, while the neutral-loss triggered MS3 approach resulted in much higher specificity. Both techniques offer a viable alternative to ETD for identifying glycated peptides when that method is unavailable.
2008. "A Perspective on the Maillard Reaction and the Analysis of Protein Glycation by Mass Spectrometry: Probing the Pathogenesis of Chronic Disease." Journal of Proteome Research 8(2):754-769. doi:DOI: 10.1021/pr800858h Abstract The Maillard reaction, starting from the glycation of protein and progressing to the formation of advanced glycation end-products (AGEs), is implicated in the development of complications of diabetes mellitus, as well as in the pathogenesis of cardiovascular, renal, and neurodegenerative diseases. In this perspective review, we provide on overview on the relevance of the Maillard reaction in the pathogenesis of chronic disease and discuss traditional approaches and recent developments in the analysis of glycated proteins by mass spectrometry. We propose that proteomics approaches, particularly bottom-up proteomics, will play a significant role in analyses of clinical samples leading to the identification of new markers of disease development and progression.
2008. "The Alpha Project: a model system for systems biology research." IET Systems Biology 2(5):222-233. doi:10.1049/iet-syb:20080127 Abstract One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling systems. In particular, the authors have begun to interpret system behaviours and their underlying molecular mechanisms through the lens of information transmission, a principal function of signalling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviours and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. The authors’ experience may provide a productive template for systems biology investigations of other cellular systems.
2008. "The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle." Proceedings of the National Academy of Sciences of the United States of America 105(39):15094-15099. doi:10.1073/pnas.0805418105 Abstract Cyanobacteria are oxygenic photosynthetic bacteria that have significant roles in global biological carbon sequestration and oxygen production. They occupy a diverse range of habitats, from open ocean, to hot springs, deserts, and arctic waters. Cyanobacteria are known as the progenitors of the chloroplasts of plants and algae, and are the simplest known organisms to exhibit circadian behavior4. Cyanothece sp. ATCC 51142 is a unicellular marine cyanobacterium capable of N2-fixation, a process that is biochemically incompatible with oxygenic photosynthesis. To resolve this problem, Cyanothece performs photosynthesis during the day and nitrogen fixation at night, thus temporally separating these processes in the same cell. The genome of Cyanothece 51142 was completely sequenced and found to contain a unique arrangement of one large circular chromosome, four small plasmids, and one linear chromosome, the first report of such a linear element in a photosynthetic bacterium. Annotation of the Cyanothece genome was aided by the use of highthroughput proteomics data, enabling the reclassification of 25% of the proteins with no informative sequence homology. Phylogenetic analysis suggests that nitrogen fixation is an ancient process that arose early in evolution and has subsequently been lost in many cyanobacterial strains. In cyanobacterial cells, the circadian clock influences numerous processes, including carbohydrate synthesis, nitrogen fixation, photosynthesis, respiration, and the cell division cycle. During a diurnal period, Cyanothece cells actively accumulate and degrade different storage inclusion bodies for the products of photosynthesis and N2-fixation. This ability to utilize metabolic compartmentalization and energy storage makes Cyanothece an ideal system for bioenergy research, as well as studies of how a unicellular organism balances multiple, often incompatible, processes in the same cell.
2008. "High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex." Journal of Biological Chemistry 283(41):27829-27837. doi:10.1074/jbc.M803918200 Abstract Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of the photosynthetic electron transport chain in plants, algae, and cyanobacteria. Utilizing a high-throughput proteomic analysis of isolated PSII complexes from the cyanobacterium Synechocystis sp. PCC 6803, we have identified four PSII associated proteins that are encoded by the cofactor integration operon (cio). This operon contains genes with putative binding domains for chlorophyll, iron-sulfur centers, and bilins. Protein levels of this operon are more abundant in several PSII lumenal mutants, suggesting an accumulation of cio products in partially assembled PSII complexes. This provides a rare example of a bacterial operon whose protein products are translationally coordinated and associated with a single protein complex. Genetic deletion of cio results in decreased oxygen evolution by PSII, suggesting that cio products may function as regulators of PSII complex assembly or degradation, maybe facilitating an uncharacterized step in PSII assembly.
2008. "Characterization of Strategies for Obtaining Confident Identifications in Bottom-Up Proteomics Measurements Using Hybrid FTMS instruments ." Analytical Chemistry 80(22):8514-8525. doi:10.1021/ac801376g Abstract Hybrid FTMS instruments, such as the LTQ-FTTM and LTQ-OrbitrapTM, are capable of generating fast duty cycle linear ion trap MS/MS data along with high resolution information without compromising the overall throughput of measurements. Combined with online LC separations, these instruments represent powerful and flexible tools for proteomics research. In the present work, we explore strategies for high throughput, high coverage proteomics measurements using hybrid FTMS instruments. Our accurate mass and time tag (AMT tag) strategy enables identification of thousands of peptides in a single LC-FTMS analysis by comparing accurate molecular mass and LC elution time information from the analysis to a reference database. An alternative strategy considered here employs linear ion trap (low resolution) MS/MS identifications generated by an appropriate search engine, such as SEQUEST; the high resolution precursor ion spectra were used to refine the MS/MS identifications, an approach termed Accurate Precursor Mass Filter (APMF). The APMF results can be additionally filtered using the LC elution time information from the AMT tag database, which constitutes a Precursor Mass and Time Filter (PMTF), the third approach implemented in this study. Both the APMF and the PMTF approaches are evaluated for coverage and confidence of peptide identifications and contrasted with the current AMT tag strategy. Two separate methodologies were used to reliably quantify identification confidence: a commonly used decoy database method and an alternative method based on the mass accuracy histogram. The two methodologies produced consistent results, confirming the validity of the identification confidence evaluations. Comparison of the three approaches has shown that the AMT tag data analysis approach may be preferential for studies giving a priority to the highest achievable coverage. The APMF approach by itself does not require AMT tag database and provides a moderate coverage combined with acceptable confidence values of ~99%.The PMTF approach yielded a significantly better peptide identification confidence, >99.9%, that essentially excluded any false peptide identifications. The results suggest that even with a perfect peptide ID (0% FDR in the peptide MS/MS database), the peak matching FDR is a function of the database size, so smaller high confidence databases are the goal. Thus a combined strategy can implement multi-pass APMF approach to generate high confidence AMT tag databases, which can be then validated using PMTF approach; the compact high quality databases will be used for subsequent high-throughput, high coverage AMT tag studies.
2008. " Trapped-ion cell with improved DC potential harmonicity for FT-ICR MS." Journal of the American Society for Mass Spectrometry 19(4):586 - 597. doi:10.1016/j.jasms.2008.01.006 Abstract The trapped-ion cell is a key component critical for optimal performance in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). We have upgraded our 12 Tesla FT-ICR instrument with a new open cylindrical cell that includes four additional cylindrical segments that serve as compensation electrodes. The DC potential on the additional segments can be set to specific pre-calculated values to suppress DC trapping field anharmonicity, in an effort to improve coherence of the ion cyclotron motion and minimize deviations from the calibration function of the ideal cell. Alternatively, the compensation potentials can be set equal to potentials of adjacent cell electrodes, which creates a DC potential distribution equivalent to that of a regular open cylindrical cell. The initial experimental characterization of both the compensated and open cell configurations was performed using ESI direct infusion of a peptide mixture. Operating the compensated cell at increased post-excitation radii resulted in improved mass measurement accuracy together with increased signal intensity, while the regular configuration exhibited peak splitting and reduced signal life time under these operating conditions. The observed improvement of the compensated cell performance was consistent with the expected behavior due to the improved DC potential harmonicity. These results confirm that the trapping DC potential harmonicity is significant for optimizing FT-ICR MS performance.
2008. "Proteomic Analysis of Stationary Phase in the Marine Bacterium 'Candidatus Pelagibacter ubique'." Applied and Environmental Microbiology 74(13):4091-4100. doi:10.1128/AEM.00599-08 Abstract Candidatus Pelagibacter ubique, an abundant marine alphaproteobacterium, subsists in nature at low ambient nutrient concentrations and may often be exposed to nutrient limitation, but its genome revealed no evidence of global regulatory adaptations to stationary phase. We used high-resolution capillary liquid chromatography (LC) coupled online to an LTQ mass spectrometer to build an Accurate Mass and Time (AMT) tag library, and employed the AMT tag approach to quantitatively examine proteome differences between exponentially growing and stationary phase Cand. P. ubique cells cultivated in a seawater medium. The AMT tag library represented 72% of the predicted protein coding genes. Stationary phase protein abundance increased for OsmC, which mitigates oxidative damage, and for molecular chaperones, enzymes involved in methionine and cysteine biosynthesis, proteins involved in rho-dependent transcription termination, and the signal transduction enzymes CheY-FisH and ChvG. Our findings indicate that Cand. P. ubique responds adaptively to stationary phase by increasing the abundance of a suite of proteins that contribute to homeostasis, but does not undergo major proteome remodeling. We speculate that this limited response may enable Cand. P. ubique to cope with ambient conditions in which nutrients are often insufficient for short periods, and the ability to resume growth overrides the capacity for long term survival afforded by more comprehensive global stationary phase responses.
2008. "Optimum Waveforms for Differential Ion Mobility Spectrometry (FAIMS)." Journal of the American Society for Mass Spectrometry 19(9):1286-1295. Abstract Differential mobility spectrometry or field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as a major tool for separation and identification of gas-phase ions, particularly in conjunction with mass-spectrometry. In FAIMS, ions are filtered by the difference between mobilities in gases (K) at high and low electric field intensity (E) using asymmetric waveforms. An infinite number of waveform profiles is possible and maximizing the performance within engineering constraints is a major issue in FAIMS technology development. Earlier optimizations assumed the non-constant component of mobility to scale as E2, producing the same result for all ions. Here we show that the optimum profiles for either rectangular or sinusoidal-based waveforms are controlled by the full series expansion of K(E) that always includes terms beyond the 1st proportional to E2. For many ion/gas pairs, the first two terms have different signs and the optimum profiles at sufficiently high E in FAIMS may substantially differ from those found previously, improving the resolving power by up to 2 - 2.5 times. This situation arises for some ions in all FAIMS systems, but becomes more common with recent miniaturized units that employ higher E. With realistic K(E) dependences, the maximum waveform amplitude is not necessarily best and reducing it by up to 20 - 30% is beneficial in some cases. Present findings are particularly relevant to targeted analyses where separation depends on the spread between K(E) functions of specific ions.
2008. " Fundamentals of Traveling Wave Ion Mobility Spectrometry." Analytical Chemistry 80(24):9689-9699. doi:10.1021/ac8016295 Abstract Traveling-wave ion mobility spectrometry (TW IMS) is a new method for IMS analyses implemented in the Synapt IMS/time-of-flight mass spectrometry system. Despite wide adoption of this platform, the fundamentals of TW IMS were understood only qualitatively and factors governing the ion transit time (the separation parameter) and resolution have remained murky. Here we develop the basic theory of TW IMS using first-principles derivations and ion dynamics simulations. The key parameter is the ratio (c) of ion drift velocity at the wave slope to wave speed (s). At low c, the ion transit velocity is proportional to the squares of mobility (K) and electric field intensity (E), as opposed to linear scaling in drift tube (DT) IMS and differential mobility analyzers (DMA). At higher c, the scaling deviates from quadratic in a manner dependent on the waveform profile, becoming more gradual with the idealized triangular profile but first steeper and then more gradual for realistic profiles with variable E. At highest c, the transit velocity asymptotically approaches the wave speed. Unlike with DT IMS, the resolving power of TW IMS depends on mobility, scaling as K1/2 in the low-c limit and slower at higher c. A nonlinear dependence of the transit time on mobility means that the true resolving power of TW IMS differs from the apparent value, often substantially. In the result, a near-optimum resolution is achievable over a ~300 - 400% range of ion mobilities. The major predicted trends are in agreement with TW IMS measurements for peptide ions as a function of ion mobility, wave amplitude, and gas pressure. The issues of proper TW IMS calibration and ion distortion by field heating are also discussed. Quantitative understanding of TW IMS separations gained in this work will allow rational optimization of instrument design and operation, and improved calibration of spectra.
2008. "Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags." Analytical Chemistry 80(6):1871-82. doi:10.1021/ac702328x Abstract Identifying proteins correctly and with known levels of confidence remain as significant challenges for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR, and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and de novo-style data analyses of high precision MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3,132 confident peptide identifications (~5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3,359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed ~5% of the 3,359 identifications to be incorrect, and many more as potentially ambiguous, (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including non-tryptic peptides, peptides with various expected/unexpected chemical modifications, known/unknown posttranslational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.
2008. "Mass Spectrometry Analysis of Proteome-wide Proteolytic Post-translational Degradation of Proteins." Analytical Chemistry 80(15):5819-5828. doi:10.1021/ac800077w Abstract Protein proteolysis is an essential component to proper cell function. Here, we demonstrate a method for studying protein degradation by detection of intermediate intracellular peptides with a high-precision tandem mass spectrometry de novo sequencing-based approach. From a Saccharomyces cerevisiae lysate, we identified >1,200 peptides containing 6-100 amino acids without random false positives and ascribed most identifications as being products of protein degradation. Most protein degradation observed was located in the cytoplasm, and multiple types of cleavage were found to exist in addition to the expected trypsin-like and chymotrypsin-like preferences. The yeast nucleus was found as a proteolysis-inert organelle under the conditions studied and the V-ATPase to be degraded during disassembly. Additionally, matrix associated mitochondrial proteins functioning as transport carriers and gates were found to be commonly degraded. Determining these protein degradation events could eventually aid in understanding of cell biology and detection and treatment of protein degradation-related diseases.
2008. "De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins ." Analytical Chemistry 80(20):7742-7754. doi:10.1021/ac801123p Abstract De novo sequencing has a promise to discover the protein post-translation modifications; however, such approach is still in their infancy and not widely applied for proteomics practices due to its limited reliability. In this work, we describe a de novo sequencing approach for discovery of protein modifications through identification of the UStags (Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry for peptides and polypeptides in a yeast lysate, and the de novo sequences obtained were filtered to define a more limited set of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags’ prefix and suffix sequences and the UStags themselves) were used to infer the possible sequence modifications. With this de novo-UStag approach, we uncovered some unexpected variances of yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. Random matching of the de novo sequences to the predicted sequences were examined with use of two random (false) databases, and ~3% false discovery rates were estimated for the de novo-UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity are described. The de novo-UStag complements the UStag method previously reported by enabling discovery of new protein modifications.
2008. "Use of high-throughput mass spectrometry to elucidate host pathogen interactions in Salmonella." Future Microbiology 3(6):625-634. Abstract Capabilities in mass spectrometry are evolving rapidly, with recent improvements in sensitivity, data analysis, and most important, from the standpoint of this review, much higher throughput allowing analysis of many samples in a single day. This short review describes how these improvements in mass spectrometry can be used to dissect host-pathogen interactions using Salmonella as a model system. This approach enabled direct identification of the majority of annotated Salmonella proteins, quantitation of expression changes under various in vitro growth conditions, and new insights into virulence and expression of Salmonella proteins within host cell cells. One of the most significant findings is that a very high percentage of the all annotated genes (>20%) in Salmonella are regulated post-transcriptionally. In addition, new and unexpected interactions have been identified for several Salmonella virulence regulators that involve protein-protein interactions, suggesting additional functions of these regulators in coordinating virulence expression. Overall high throughput mass spectrometry provides a new view of pathogen-host interactions emphasizing the protein products and defining how protein interactions determine the outcome of infection.
2008. "Control of Ion Distortion in Field Asymmetric Waveform Ion Mobility Spectrometry via Variation of Dispersion Field and Gas Temperature ." Analytical Chemistry 80(19):7508-7515. doi:10.1021/ac800655d Abstract Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as an analytical tool of broad utility, especially in conjunction with mass spectrometry. Of particular promise is the use of FAIMS and 2-D ion mobility methods that combine it with conventional IMS to resolve and characterize protein and other macromolecular conformers. However, FAIMS operation requires high electric fields and ions are inevitably heated by above-thermal collisions with buffer gas molecules. This may induce ion isomerization and dissociation that distort separation properties determined by FAIMS and subsequent stages and/or reduce instrumental sensitivity. As FAIMS employs a periodic waveform, the ion temperature can be characterized at the maximum or average field intensity (E). Which method is most applicable to temperature sensitive ions, such as protein ions, has been debated. Here we address this issue by measuring the unfolding of compact ubiquitin ion geometries as a function of waveform amplitude (dispersion field, ED) and gas temperature, T. The field heating is quantified by matching the dependences of structural transitions on ED and T. Increasing ED from 12 to 16 or from 16 to 20 kV/cm is equivalent to heating the (N2) gas by ~15 - 25 oC. The magnitude of field heating for any E can be calculated using the two-temperature theory, and raising ED by 4 kV/cm augments heating by ~15 - 30 oC for maximum and ~4 - 8 oC for average E in the FAIMS cycle. Hence, isomerization of ions in FAIMS appears to be governed by the maximum internal temperature at waveform peaks.
2008. "Enhanced Detection of Low Abundant Human Plasma Proteins using a Tandem IgY12-SuperMix Immunoaffinity Separation Strategy." Molecular & Cellular Proteomics. MCP 7(10):1963-1973. doi:10.1074/mcp.M800008-MCP200 Abstract The enormous dynamic range of human bodily fluid proteomes poses a significant challenge for current MS-based proteomic technologies as it makes it especially difficult to detect low-abundant proteins in human biofluids such as blood plasma, an essential aspect for successful biomarker discovery efforts. Here we present a novel tandem IgY12-SuperMix immunoaffinity separation system for enhanced detection of low-abundant proteins in human plasma. The tandem IgY12-SuperMix system separates ~60 abundant proteins from those low abundant proteins in plasma, thus allowing significant enrichment of low-abundance plasma proteins in the flow-through fraction. High reproducibility of the tandem separations in both sample processing recovery and LC-MS/MS identification results were observed, comparable to the results from the single IgY12 separations. Tandem SuperMix flow-through samples based on single dimensional (1D) and two dimensional (2D) LC-MS/MS analyses revealed a 60-80% improvement in overall proteome coverage when compared to the single IgY12 flow-through, suggesting significantly enhanced detection of low-abundant proteins. A total of 695 plasma proteins were confidently identified in a single analysis (with a minimum of two peptides per protein) by coupling the tandem separation strategy with 2D-LC-MS/MS, including 42 proteins with normal reported concentrations ranging from ~100 pg/mL to 100 ng/mL. The concentrations of two selected proteins M-CSF and MMP8 were independently validated by ELISA as 202 pg/mL and 12.4 ng/mL, respectively. Evaluation of binding efficiency revealed 45 medium-abundant proteins efficiently captured by the SuperMix column with >90% retention. Taken together, the results illustrate the potential broad utilities of this tandem IgY12-SuperMix strategy in proteomic applications involving human biofluids where effectively addressing the dynamic range of the specimen is imperative.
2008. "Characterization of the mouse bronchoalveolar lavage proteome by micro-capillary LC–FTICR mass spectrometry." Journal of Chromatography B 864(1-2):95-101. Abstract Bronchoalveolar lavage fluid (BALF) contains proteins derived from various pulmonary cell types, secretions and blood. As the characterization of the BALF proteome will be instrumental in establishing potential biomarkers of pathophysiology in the lungs, the objective of this study was to contribute to the comprehensive collection of Mus musculus BALF proteins using high resolution and highly sensitive micro-capillary liquid chromatography (microLC) combined with state-of-the-art high resolution mass spectrometry (MS). BALF was collected from ICR and C57BL/6 male mice exposed to nose-only inhalation to either air or cigarette smoke. The tandem mass spectra were analyzed by SEQUEST for peptide identifications with the subsequent application of accurate mass and time tags resulting in the identification of 1797 peptides with high confidence by high resolution MS. These peptides covered 959 individual proteins constituting the largest collection of BALF proteins to date. High throughput monitoring profiles of this extensive collection of BALF proteins will facilitate the discovery and validation of biomarkers that would elucidate pathogenic or adaptive responses of the lungs upon toxic insults.
2008. " DAnTE: a statistical tool for quantitative analysis of –omics data." Bioinformatics 24(13):1556-1558. doi:10.1093/bioinformatics/btn217 Abstract DAnTE (Data Analysis Tool Extension) is a statistical tool designed to address challenges unique to quantitative bottom-up, shotgun proteomics data. This tool has also been demonstrated for microarray data and can easily be extended to other high-throughput data types. DAnTE features selected normalization methods, missing value imputation algorithms, peptide to protein rollup methods, an extensive array of plotting functions, and a comprehensive ANOVA scheme that can handle unbalanced data and random effects. The Graphical User Interface (GUI) is designed to be very intuitive and user friendly.
2008. "Elimination of Systematic Mass Measurement Errors in Liquid Chromatography-Mass Spectrometry Based Proteomics using Regression Models and a priori Partial Knowledge of the Sample Content ." Analytical Chemistry 80(3):693-706. doi:10.1021/ac701863d Abstract The high mass measurement accuracy and precision available with recently developed mass spectrometers is increasingly used in proteomics analyses to confidently identify tryptic peptides from complex mixtures of proteins, as well as post-translational modifications and peptides from non-annotated proteins. To take full advantage of high mass measurement accuracy instruments it is necessary to limit systematic mass measurement errors. It is well known that errors in the measurement of m/z can be affected by experimental parameters that include e.g., outdated calibration coefficients, ion intensity, and temperature changes during the measurement. Traditionally, these variations have been corrected through the use of internal calibrants (well-characterized standards introduced with the sample being analyzed). In this paper we describe an alternative approach where the calibration is provided through the use of a priori knowledge of the sample being analyzed. Such an approach has previously been demonstrated based on the dependence of systematic error on m/z alone. To incorporate additional explanatory variables, we employed multidimensional, nonparametric regression models, which were evaluated using several commercially available instruments. The applied approach is shown to remove any noticeable biases from the overall mass measurement errors, and decreases the overall standard deviation of the mass measurement error distribution by 1.2- to 2-fold, depending on instrument type. Subsequent reduction of the random errors based on multiple measurements over consecutive spectra further improves accuracy and results in an overall decrease of the standard deviation by 1.8- to 3.7-fold. This new procedure will decrease the false discovery rates for peptide identifications using high accuracy mass measurements.
2008. "Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues." Journal of Proteome Research 7(8):3114-3126. doi:10.1021/pr800205b Abstract The pancreatic islets of Langerhans and insulin-producing beta cells in particular play a central role in the maintenance of glucose homeostasis and the islet dysfunction is associated with the pathogenesis of both type 1 and type 2 diabetes mellitus. To contribute to the understanding of the biology of the pancreatic islets we applied proteomic techniques based on liquid chromatography coupled with mass spectrometry. Here as an initial step we present the first comprehensive proteomic characterization of pancreas islets of the mouse, the commonly used animal model for diabetes research. Two-dimensional SCX LC/RP LC-MS/MS has been applied to characterize of the mouse islet proteome, resulting in the confident identification of 17,350 different tryptic peptides covering 2,612 proteins with at least two unique peptide identifications per protein. The dataset also allowed identification of a number of post-translational modifications including several modifications relevant to oxidative stress and phosphorylation. While many of the identified phosphorylation sites corroborates with previous known sites, the oxidative modifications observed on cysteinyl residues potentially reveal novel information related to the role of oxidation stress in islet functions. Comparative analysis of the islet proteome database with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 68 proteins uniquely detected only in the pancreatic islets. Besides proteins with known functions, like islet secreted peptide hormones, this unique set contains a number of proteins with yet unknown functions. The resulting peptide and protein database will be available at ncrr.pnl.gov web site of the NCRR proteomic center (ncrr.pnl.gov).
2008. "“Spatial Mapping of the Neurite and Soma Proteomes Reveals a Functional Cdc42/Rac Regulatory Network”." Proceedings of the National Academy of Sciences of the United States of America 105(6):1931-1936. doi:10.1073/pnas.0706545105 Abstract Neurite extension and growth cone navigation are guided by extracellular cues that control cytoskeletal rearrangements. However, understanding the complex signaling mechanisms that mediate neuritogenesis has been limited by the inability to biochemically separate the neurite and soma for spatial proteomic and bioinformatic analyses. Here, we apply global proteome profiling in combination with a novel neurite purification methodology for comparative analysis of the soma and neurite proteomes of neuroblastoma cells. The spatial relationship of 4855 proteins were mapped revealing networks of signaling proteins that control integrins, the actin cytoskeleton, and axonal guidance in the extending neurite. Bioinformatics and functional analyses revealed a spatially compartmentalized Rac/Cdc42 signaling network that operates in conjunction with multiple GEFs and GAPs to control neurite formation. Interestingly, RNA interference experiments revealed that the different GEFs and GAPs regulate specialized functions during neurite formation including neurite growth and retraction kinetics, cytoskeletal organization, and cell polarity. Our findings provide insight into the spatial organization of signaling networks that enable neuritogenesis and provide a comprehensive system-wide profile of proteins that mediate this process including those that control Rac and Cdc42 signaling.
2008. "Subambient pressure ionization with nanoelectrospray source and interface for improved sensitivity in mass spectrometry ." Analytical Chemistry Vol. 80(5):1800-1805. doi:10.1021/ac702354b Abstract An electrospray ionization mass spectrometry (ESI-MS) source and interface has been designed that enables efficient ion production and transmission in a 30 Torr pressure environment using solvents compatible with typical reverse-phase liquid chromatography (RPLC) separations. In this design, the electrospray emitter is located inside the mass spectrometer in the same region as an electrodynamic ion funnel. This avoids the use of a conductance limit ion inlet, as required by a conventional atmospheric pressure ESI source, and allows more efficient ion transmission to the mass analyzer. Performance of the low pressure ESI source and interface was evaluated by electrospraying standard solutions at 300 nL/min, and comparing results with those obtained from a standard atmospheric pressure ESI source that used a heated capillary inlet. The importance of desolvation was also investigated by electrospraying at different flow rates, which showed that the ion funnel provided an effective desolvation region to aid the creation of gas phase analyte ions. This initial study demonstrated a ~ 5-fold improvement in sensitivity when the low pressure ESI source was used compared to a standard atmospheric pressure ESI source.
2008. "Improving low-level plasma protein mass spectrometry-based detection for candidate biomarker discovery and validation." ICBC Newsletter 3(3):, Abstract Methods. To improve the detection of low abundance protein candidate biomarker discovery and validation, particularly in complex biological fluids such as blood plasma, increased sensitivity is desired using mass spectrometry (MS)-based instrumentation. A key current limitation on the sensitivity of electrospray ionization (ESI) MS is due to the fact that many sample molecules in solution are never ionized, and the vast majority of the ions that are created are lost during transmission from atmospheric pressure to the low pressure region of the mass analyzer. Two key technologies, multi-nanoelectrospray emitters and the electrodynamic ion funnel have recently been developed and refined at Pacific Northwest National Laboratory (PNNL) to greatly improve the ionization and transmission efficiency of ESI MS based analyses. Multi-emitter based ESI enables the flow from a single source (typically a liquid chromatography [LC] column) to be divided among an array of emitters (Figure 1). The flow rate delivered to each emitter is thus reduced, allowing the well-documented benefits of nanoelectrospray 1 for both sensitivity and quantitation to be realized for higher flow rate separations. To complement the increased ionization efficiency afforded by multi-ESI, tandem electrodynamic ion funnels have also been developed at PNNL, and shown to greatly improve ion transmission efficiency in the ion source interface.2, 3 These technologies have been integrated into a triple quadrupole mass spectrometer for multiple reaction monitoring (MRM) of probable biomarker candidates in blood plasma and show promise for the identification of new species even at low level concentrations.
2008. "MASIC: a software program for fast quantitation and flexible visualization of chromatographic profiles from detected LC-MS(/MS) features." Computational Biology and Chemistry 32(3):215-217. doi:10.1016/j.compbiolchem.2008.02.006 Abstract Quantitative analysis of liquid chromatography (LC)- mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data is essential to many proteomics studies. We have developed MASIC to accurately measure peptide abundances and LC elution times in low-resolution LC-MS/MS analyses. This software program uses an efficient processing algorithm to quickly generate mass specific selected ion chromatograms from a dataset and provides an interactive browser that allows users to examine individual chromatograms in a variety of fashions. The improved elution time estimates afforded by MASIC increase the utility of LC-MS/MS data in the accurate mass and time (AMT) tag approach to proteomics.
2008. "High Resolution Separations and Improved Ion Production and Transmission in Metabolomics." Trends in Analytical Chemistry. TrAC 27(3):205-214. doi:10.1016/j.trac.2007.11.003 Abstract The goal of metabolomics experiments is the detection and quantitation of as many sample components as reasonably possible in order to identify “features” that can be used to characterize the samples under study. When utilizing electrospray ionization to produce ions for analysis by mass spectrometry (MS), it is imperative that metabolome sample constituents be efficiently separated prior to ion production, in order to minimize the phenomenon of ionization suppression. Similarly, optimization of the MS inlet can lead to increased measurement sensitivity. This review will focus on the role of high resolution liquid chromatography (LC) separations in conjunction with improved ion production and transmission for LC-MS-based metabolomics.
2008. "Application of proteomics in the discovery of candidate protein biomarkers in a Diabetes Autoantibody Standardization Program sample subset ." Journal of Proteome Research 7(2):698-707. doi:10.1021/pr700606w Abstract Objective. Before biomarkers predictive of type 1 diabetes can be evaluated in proficiency evaluations, they must be identified and validated in initial, exploratory studies. Hypothesis-driven comparative studies may be performed to identify candidate biomarkers but are limited to the current knowledge of metabolic, signaling, and inflammatory pathways in the context of type 1 diabetes. Alternatively, untargeted “-omics” approaches may be employed in profiling studies to identify candidate biomarkers of type 1 diabetes.
2008. "DeconMSn: A Software Tool for accurate parent ion monoisotopic mass determination for tandem mass spectra." Bioinformatics 24(7):1021-1023. doi:10.1093/bioinformatics/btn063 Abstract We present a new software tool for tandem MS analyses that: • accurately calculates the monoisotopic mass and charge of high–resolution parent ions • accurately operates regardless of the mass selected for fragmentation • performs independent of instrument settings • enables optimal selection of search mass tolerance for high mass accuracy experiments • is open source and thus can be tailored to individual needs • incorporates a SVM-based charge detection algorithm for analyzing low resolution tandem MS spectra • creates multiple output data formats (.dta, .MGF) • handles .RAW files and .mzXML formats • compatible with SEQUEST, MASCOT, X!Tandem
2008. "Human Proteinpedia enables sharing of human protein data." Nature Biotechnology 26(2):164-167. Abstract Proteomic technologies, such as yeast twohybrid, mass spectrometry (MS), protein/ peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories. Even more difficult is the annotation and display of pertinent information in the context of the corresponding proteins. Wikipedia, an online encyclopedia that anyone can edit, has already proven quite successful1 and can be used as a model for sharing biological data. However, the need for experimental evidence, data standardization and ownership of data creates scientific obstacles.
2008. "Analytical Characterization of the Electrospray Ion Source in the Nanoflow Regime." Analytical Chemistry 80(17):6573-6579. doi:10.1021/ac800683s Abstract We provide a thorough characterization of the low-flow electrospray as an ionization source for mass spectrometry (MS) using solutions typical for reversed-phase liquid chromatography. As expected, the electrospray operating regime strongly affects the MS signal; however, contrary to conventional wisdom, the pulsating regime consistently offers better performance than the cone-jet regime in these experimental conditions. We explain this observation by a highly efficient ionization achieved by the pulsating electrospray at low flow rates, rendering the increased charge generated by a cone-jet electrospray detrimental for transmission from atmospheric pressure to vacuum through a heated capillary interface. Over a wide range of voltages, the pulsating electrospray provides a relatively constant MS signal intensity, which depends significantly on the distance between the emitter and the MS inlet. For cone-jet electrosprays the MS signal decreases slightly with increasing voltage, but the signal is less affected by the emitter-inlet distance. At flow rates up to 100 nL/min the MS signal increases with increasing flow rate due to the larger number of ions supplied into the gas phase. At flow rates greater than 100 nL/min, the signal reaches a plateau due to increasingly unsatisfactory ionization efficiency at larger flow rates.
2008. "Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions." Journal of Proteome Research 3:960-8. doi:10.1021/pr070432+ Abstract Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.
2008. "Rapid Sample Processing For LC-MS Based Quantitative Proteomics Using High Intensity Focused Ultrasounds." Journal of Proteome Research 7(9):3860-3867. doi:10.1021/pr800161x Abstract A new sample processing workflow that uses high intensity focused ultrasound to rapidly reduce and alkylate cysteines, digest proteins and then label peptides with 18O was developed for quantitative proteomics applications. Each step was individually refined to minimize reaction times, peptide loses and undesired by-products or modifications. By using this novel workflow, mouse plasma proteins were successfully denatured, alkylated, in-solution digested, and 18O labelled in < 10 min for subsequent analysis by liquid chromatography-electrospray ionization high resolution mass spectrometry. Performance was evaluated in terms of the number of mouse plasma peptides and proteins identified in a shotgun approach and the quantitative dynamic range. The results were compared with previously published results obtained using conventional sample preparation methods and were found to be similar. Advantages of the new method include greatly simplified and accelerated sample processing, as well as being readily amenable to automation.
2008. "Application of pressurized solvents for ultrafast trypsin hydrolysis in proteomics: Proteomics on the fly." Journal of Proteome Research 7(8):3276-3281. doi:10.1021/pr7008077 Abstract A new method for rapid proteolytic digestion of proteins under high pressure that uses pressure cycling technology in the range of 5 to 35 kpsi was demonstrated for proteomic analysis. Successful in-solution digestions of single proteins and complex protein mixtures were achieved in 60 s and then analyzed by reversed phase liquid chromatography-electrospray ionization ion trap-mass spectrometry. Method performance in terms of the number of Shewanella oneidensis peptides and proteins identified in a shotgun approach was evaluated relative to a traditional “overnight” sample preparation method. Advantages of the new method include greatly simplified sample processing, easy implementation, no cross contamination among samples, and cost effectiveness.
2008. "Fully automated four-column capillary LC-MS system for maximizing throughput in proteomic analyses." Analytical Chemistry 80(1):294-302. doi:10.1021/ac701727r Abstract We describe a 4-column, high-pressure capillary liquid chromatography (LC) system for robust, high-throughput LC-MS(/MS) analyses. This system performs multiple LC separations in parallel, but staggers each of them such that the data-rich region of each separation is sampled sequentially. By allowing nearly continuous data acquisition, this design maximizes the use of the mass spectrometer. Each analytical column is connected to a corresponding ESI emitter in order to avoid the use of post-column switching and associated dead volume issues. Encoding translation stages are employed to sequentially position the emitters at the MS inlet. The high reproducibility of this system is demonstrated using consecutive analyses of global tryptic digest of the microbe Shewanella oneidensis.
2008. "Post-translational Modifications of Chicken Myelin Basic Protein Charge Components." Neurochemical Research 34(2):360-372. doi:10.1007/s11064-008-9788-4 Abstract Purified myelin basic protein (MBP) from various species contains several post-translationally modified forms termed charge components or charge isomers. Chicken MBP contains four charge components denoted as C1, C2, C3 and C8. (The C8 isomer is a complex mixture and was not investigated in this study.) These findings are in contrast to those found for human, bovine and other mammalian MBP’s. Mammalian MBP’s, each of which contain seven or eight charge components depending on the analysis of the CM-52 chromatographic curves and the PAGE gels obtained under basic pH conditions. Chicken MBP components C1, C2 and C3 were treated with trypsin and endoproteinase Glu-C. The resulting digests were analyzed by capillary liquid chromatography combined with either an ion trap tandem mass spectrometer or with a Fourier transform ion cyclotron resonance mass spectrometer. This instrumentation permitted establishing the amino acid composition and the determination of the posttranslational modifications for each of the three charge components C1-C3. With the exception of N-terminal acetylation, the post-translational modifications were partial.
2008. "Nanoelectrospray Emitter Arrays Providing Interemitter Electric Field Uniformity." Analytical Chemistry 80(14):5660-5665. doi:10.1021/ac800508q Abstract Arrays of electrospray ionization (ESI) emitters have been reported previously as a means of achieving the enhanced ionization efficiencies. A key challenge when working with multiple, closely spaced ESI emitters is overcoming the deleterious effects caused by electrical interference among neighboring emitters. Individual emitters experience different electric fields depending on their relative position in the array, such that it becomes difficult to operate all of the emitters optimally for a given applied potential. In this work, we have developed multi-nanoESI emitters arranged with a circular pattern, which enable the constituent emitters to experience a uniform electric field. The performance of the circular emitter array was compared to a single emitter and to a previously developed linear emitter array, which verified that improved electric field uniformity was achieved with the circular arrangement. The circular arrays were also interfaced with a mass spectrometer via a matching multi-capillary inlet, and the results were compared with those obtained using a single emitter. By minimizing inter-emitter electric field inhomogeneities, much larger arrays having closer emitter spacing should be feasible.
2008. "Elastomeric Microchip Electrospray Emitter for Stable Cone-Jet Mode Operation in the Nanoflow Regime." Analytical Chemistry 80(10):3824-3831. Abstract Despite widespread interest in applying lab-on-a-chip technologies to mass spectrometry (MS)-based analyses, the coupling of microfluidics to electrospray ionization (ESI)-MS remains challenging. We report a robust, integrated poly(dimethylsiloxane) microchip interface for ESI-MS using simple and widely accessible microfabrication procedures. The interface uses an auxiliary channel to provide electrical contact in the Taylor cone of the electrospray without sample loss or dilution. The electric field at the channel terminus is enhanced by two vertical cuts that cause the interface to taper to a line rather than to a point, and the formation of small Taylor cones at the channel exit ensures sub-nL post-column dead volumes. While comparable ESI-MS sensitivities were achieved using both microchip and conventional fused silica capillary emitters, stable cone-jet mode electrospray could be established over a far broader range of flow rates (from 50–1000 nL/min) and applied potentials using the microchip emitters. This special feature of the microchip emitter should minimize the fine tuning required for electrospray optimization and make the stable electrospray more resistant to external perturbations.
2008. "Capillary-Based Multi Nanoelectrospray Emitters: Improvements in Ion Transmission Efficiency and Implementation with Capillary Reversed-Phase LC-ESI-MS." Analytical Chemistry 80(1):143-149. doi:10.1021/ac701647s Abstract We describe the coupling of liquid chromatography (LC) separations with mass spectrometry (MS) using nanoelectrospray ionization (nanoESI) multi-emitters. The array of 19 emitters reduced the flow rate delivered to each emitter, allowing the enhanced sensitivity that is characteristic of nanoESI to be extended to higher flow rate separations. The signal for peptides from spiked proteins in a human plasma tryptic digest increased 11-fold on average when the multi-emitters were employed, due to increased ionization efficiency and improved ion transfer efficiency through a newly designed heated multi-capillary MS inlet. Additionally, the LC peak signal-to-noise ratio increased ~7-fold when the multi-emitter configuration was used. The low dead volume of the emitter arrays preserved peak shape and resolution for robust capillary LC separations using total flow rates of 2-µL/min.
2008. "The Mammary Epithelial Cell Secretome and its Regulation by Signal Transduction Pathways." Journal of Proteome Research 7(2):558-569. doi:10.1021/pr0704377 Abstract Extracellular proteins released by mammary epithelial cells are critical mediators of cell communication, proliferation and organization, yet the actual spectrum of proteins released by any given cell (the secretome) is poorly characterized. To define the set of proteins secreted by human mammary epithelial cells (HMEC), we combined analytical and computational approaches to define a secretome protein set based upon probable biological significance. Analysis of HMEC-conditioned medium by liquid chromatography-mass spectrometry resulted in identification of 889 unique proteins, of which 151 were found to be specifically enriched in the extracellular compartment when compared with a database of proteins expressed in whole HMEC lysates. Additional high mass accuracy analysis revealed 36 proteins whose extracellular abundance increased after treatment with phorbol ester (PMA), a protein kinase C agonist and general secretagogue. Many of the PMA stimulated proteins have been reported to be aberrantly expressed in human cancers and appear to be co-regulated as multigene clusters. By inhibiting PMA-mediated transactivation of the epidermal growth factor receptor (EGFR), a pathway critically required for normal HMEC function, we found that the secretion of specific matrix metalloproteases were also coordinately regulated through EGFR transactivation. This study demonstrates a tiered strategy by which extracellular proteins can be identified and progressively assigned to classes of increasing confidence and regulatory importance.
2008. "Automated Gain Control Ion Funnel Trap for Orthogonal Time-of-Flight Mass Spectrometry ." Analytical Chemistry 80(14):5367-5376. doi:10.1021/ac8003488 Abstract Time-of-flight mass spectrometry (TOF MS) is increasingly used in proteomics research. Herein, we report on the development and characterization of an ultra-sensitive TOF MS instrument equipped with an electrodynamic ion funnel trap (IFT) that employs an automatic gain control (AGC) capability. The IFT-TOF MS was coupled to a reverse-phase capillary liquid chromatography (RPLC) separation and evaluated in experiments with complex proteolytic digests. When applied to a global tryptic digest of Shewanella oneidensis proteins, an order-of-magnitude increase in sensitivity compared to that of the conventional continuous mode of operation was achieved due to efficient ion accumulation prior to TOF MS analysis. As a result of this sensitivity improvement and related improvement in mass measurement accuracy, the number of unique peptides identified in the AGC-IFT mode was 5-fold greater than that obtained in the continuous mode.
2008. "The Influence of Sample Preparation and Replicate Analyses on HeLa Cell Phosphoproteome Coverage." Journal of Proteome Research 7(6):2215-2221. Abstract Ongoing optimization of proteomic methodologies seeks to improve both coverage and confidence in protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample) and biological replicates (multiple individual samples) is crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 uniquely identified peptides per dataset) affords the optimal collection of peptide information.
2008. "Comparative proteogenomics: combining mass spectrometry and comparative genomics to analyze multiple genomes." Genome Research 18(7):1133-1142. Abstract While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages and cleavage of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.
2008. "Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing." Journal of Virology 82(13):6524-6535. doi:doi:10.1128/JVI.00502-08 Abstract Human cytomegalovirus (HCMV) is implicated in the acceleration of a number of vascular diseases including transplant vascular sclerosis (TVS), the lesion associated with chronic rejection (CR) of solid organ transplants. Although the virus persists in the allograft throughout the course of disease, few cells are directly infected by CMV. This observation is in contrast to the global effects that CMV has on the acceleration of TVS/CR, suggesting that CMV infection indirectly promotes the vascular disease process. Recent transcriptome analysis of CMV-infected heart allografts indicates that the virus induces cytokines and growth factors associated with angiogenesis (AG) and wound healing (WH), suggesting that CMV may accelerate TVS/CR through the induction and secretion of AG/WH factors from infected cells. We analyzed virus-free supernatants from HCMV-infected cells (HCMV secretomes) for growth factors, by mass spectrometry and immunoassays, and found that the HCMV secretome contains over 1,000 cellular proteins, many of which are involved in AG/WH. Importantly, functional assays demonstrated that CMV but not herpes simplex virus secretomes not only induce AG/WH but also promote neovessel stabilization and endothelial cell survival for 2 weeks. These findings suggest that CMV acceleration of TVS occurs through virus-induced growth factors and cytokines in the CMV secretome.
2008. "Linear Discriminant Analysis - Based Estimation of the False Discovery Rate for Phosphopeptide Identifications ." Journal of Proteome Research 7(6):2195-2203. doi:10.1021/pr070510t Abstract This paper describes a method to estimate the False Discovery Rate (FDR) of phosphopeptide identifications. The method starts with a re-assignment of the phosphorylation site/sites to those phosphopeptides for which there exists an ambiguity in the original assignment of the phosphorylation site/sites. It then performs an online data training using Expectation Maximization to estimate the joint distribution underlying the observed search results of multiple parameters from search engines. A Linear Discriminant Analysis (LDA) is subsequently carried out to optimally combine the search results into a discriminant score that possesses the most discriminating power. Based on the discriminant score, the p-value and q-value (explain a little bit more) for each identified phosphopeptide are calculated and the FDR for the set of phosphopeptides which are claimed correct identifications can then be rigorously estimated based on its definition. The approach can be easily extended to estimate the FDR of unmodified peptides. The proposed approach has been applied to datasets from a study of the effect of high-dose radiation on human skin fibroblast cells. The data analysis procedure has been coded into a software package which is freely available.
2008. "A Computational Strategy to Analyze Label-Free Temporal Bottom-up Proteomics Data." Journal of Proteome Research 7(7):2595-2604. doi:10.1021/pr0704837 Abstract Motivation: Biological systems are in a continual state of flux, which necessitates an understanding of the dynamic nature of protein abundances. The study of protein abundance dynamics has become feasible with recent improvements in mass spectrometry-based quantitative proteomics. However, a number of challenges still re-main related to how best to extract biological information from dy-namic proteomics data; for example, challenges related to extrane-ous variability, missing abundance values, and the identification of significant temporal patterns. Results: This article describes a strategy that addresses the afore-mentioned issues for the analysis of temporal bottom-up proteomics data. The core strategy for the data analysis algorithms and subse-quent data interpretation was formulated to take advantage of the temporal properties of the data. The analysis procedure presented herein was applied to data from a Rhodobacter sphaeroides 2.4.1 time-course study. The results were in close agreement with existing knowledge about R. sphaeroides, therefore demonstrating the utility of this analytical strategy.
2008. "Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor." Biochimica et Biophysica Acta--Proteins and Proteomics 1784(12):1935-1941. doi:10.1016/j.bbapap.2008.06.011 Abstract e(III) oxides are the most abundant source of reducible Fe(III) by microorganisms in most soils and sediments, yet few studies on the physiology of Fe(III)-reducing microorganisms during growth on Fe(III) oxide have been conducted because of the technical difficulties in working with cell growth and harvest in the presence of Fe(III) oxides. Geobacter sulfurreducens is a representative of the Geobacter species that predominate in a variety of subsurface environments in which Fe(III) oxide is important. In order to better understand the physiology of Geobacter species during growth on Fe(III) oxide, the proteome of G. sulfurreducens grown on Fe(III) oxide was compared with the proteome of cells grown with soluble Fe(III) citrate. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) revealed 19 proteins that were more abundant during growth on Fe(III) oxide than on soluble Fe(III). These included proteins related to protein synthesis, electron transfer and energy production, oxidative stress, protein folding, outer membrane proteins, nitrogen metabolism and hypothetical proteins. Further analysis of the proteome with the accurate mass and time (AMT) tag method revealed additional proteins associated with growth on Fe(III) oxide. These included the outer-membrane c-type cytochrome, OmcS and OmcG, which genetic studies have suggested are required for Fe(III) oxide reduction. Furthermore, several other cytochromes, as yet unstudied, were detected to be significantly up regulated during growth on Fe(III) oxide and other proteins of unknown function were more abundant during growth on Fe(III) oxide than on soluble Fe(III). PilA, the structural protein for pili, which is required for Fe(III) oxide reduction, and other pilin-associated proteins were also more abundant during growth on Fe(III) oxide. Confirmation of the differential expression of proteins known to be important in Fe(III) oxide reduction was observed, and an additional number of previously unidentified proteins were found with significant abundance in the cells grown under conditions of Fe(III) oxide reduction.
2008. "Application of the accurate mass and time tag approach in studies of the human blood lipidome." Journal of Chromatography B 871(2):243-252. doi:10.1016/j.jchromb.2008.04.040 Abstract We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput quantitative LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance. In addition, we also report on the optimization of a reversed-phase LC method for the separation of lipids in these sample types.
2008. "Enhanced Ion Utilization Efficiency Using an Electrodynamic Ion Funnel Trap as an Injection Mechanism for Ion Mobility Spectrometry ." Analytical Chemistry 80(3):612-623. doi:10.1021/ac701648p Abstract Conventional ion mobility spectrometers that sample ion packets from continuous sources have traditionally been constrained by an inherently low duty cycle. As such, ion utilization efficiencies have been limited to <1% in order to maintain acceptable levels of instrumental resolving power. Using a redesigned electrodynamic ion funnel, we demonstrated the ability to accumulate, store, and eject ions in conjunction with ion mobility spectrometry (IMS), which elevated the charge density of the ion packets ejected from the ion funnel trap (IFT) and provided a considerable increase in the overall ion utilization efficiency of the IMS instrument. A 7-fold increase in signal intensity was revealed by comparing continuous ion beam current with the amplitude of the pulsed ion current in IFT-IMS experiments. In addition to reporting performance enhancements for an IMS instrument equipped with a Faraday plate, we evaluate IFT operating characteristics using a time-of-flight mass spectrometer attached to the IMS drift tube.
2008. " Pseudorandom Sequence Modifications for Ion Mobility Orthogonal Time-of-Flight Mass Spectrometry." Analytical Chemistry 80(7):2464-2473. doi:10.1021/ac7022712 Abstract Due to the inherently low duty cycle of ion mobility spectrometry (IMS) experiments that sample from continuous ion sources, a range of experimental advances have been developed to maximize ion utilization efficiency. The use of ion trapping mechanisms prior to the ion mobility drift tube has demonstrated significant gains over discrete sampling from continuous sources; however, these technologies have traditionally relied upon a signal averaging to attain analytically relevant signal-to-noise ratios (SNR). Multiplexed (MP) techniques based upon the Hadamard transform offer an alternative experimental approach by which ion utilization efficiency can be elevated to ~ 50 %. Recently, our research group demonstrated a unique multiplexed ion mobility time-of-flight (MP-IMS-TOF) approach that incorporates ion trapping and can extend ion utilization efficiency beyond 50 %. However, the spectral reconstruction of the multiplexed signal using this experiment approach requires the use of sample-specific weighing designs. Though general weighing designs have been shown to significantly enhance ion utilization efficiency using this MP technique, such weighing designs cannot be applied to all samples. By modifying both the ion funnel trap and the pseudo random sequence (PRS) used for the MP experiment we have eliminated the need for complex weighing matrices. For both simple and complex mixtures SNR enhancements of up to 13 were routinely observed as compared to the SA-IMS-TOF experiment. In addition, this new class of PRS provides a two fold enhancement in ion throughput compared to the traditional HT-IMS experiment.
2008. "Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease." Journal of Proteome Research 7(2):666-677. doi:10.1021/pr070546l Abstract The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.
2008. "Comparative Bacterial Proteomics: Analysis of the Core Genome Concept." PLoS One 3(2):e1542. Abstract Comparative bacterial genomic studies commonly predict a set of genes indicative of common ancestry. Experimental validation of the existence of this core genome requires extensive measurement and is not typically undertaken. Enabled by an extensive proteome database development over a six year period, we experimentally verified the expression of proteins predicted from genomic ortholog comparisons among 17 environmental and pathogenic bacteria. More exclusive relationships were observed among the expressed protein content of phenotypically related bacteria, which is indicative of the specific lifestyles associated with these organisms. While genomic studies establish relative orthologous relationships among a set of bacteria and propose a set of ancestral genes, our proteomics study establishes expressed lifestyle differences among conserved genes and proposes a set of expressed ancestral traits.
2008. "Optimization of proteomic sample preparation procedures for comprehensive protein characterization of pathogenic systems." Journal of Biomolecular Techniques:JBT 19(5):285-295. Abstract The elucidation of critical functional pathways employed by pathogens and hosts during an infectious cycle is both challenging and central to our understanding of infectious diseases. In recent years, mass spectrometry-based proteomics has been used as a powerful tool to identify key pathogenesis-related proteins and pathways. Despite the analytical power of mass spectrometry-based technologies, samples must be appropriately prepared to characterize the functions of interest (e.g. host-response to a pathogen or a pathogen-response to a host). The preparation of these protein samples requires multiple decisions about what aspect of infection is being studied, and it may require the isolation of either host and/or pathogen cellular material.
2008. "Dynamically Multiplexed Ion Mobility Time-of-Flight Mass Spectrometry ." Analytical Chemistry 80(15):5873-5883. doi:10.1021/ac8003665 Abstract Ion Mobility Spectrometry–Time-of-Flight Mass Spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity high-throughput platform for e.g. proteomics applications. In this work, we have developed and integrated three advanced technologies, enabling (1) efficient ion accumulation in the ion funnel trap prior to IMS separation, (2) multiplexing (MP) of ion packet introduction into the IMS drift tube and (3) signal detection with an analog-to-digital converter (ADC), into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of e.g. blood plasma. To better address variable sample complexity, we have additionally developed and rigorously evaluated a new dynamic MP approach that ensures correlation of the analyzer performance with an ion source function, and provides the improved dynamic range and sensitivity. The MP IMS-TOF MS instrument has been shown to reliably detect peptides at a concentration of 1 nM in a highly complex matrix, as well as to provide a four orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features yielded ~ 700 unique peptide identifications at a false discovery rate (FDR) of ~ 7.5 %. Accounting for IMS information gave rise to a projected FDR of ~ 4 %. Signal reproducibility was found to be greater than 80 %, while the variations in the number of unique peptide identifications were < 15 %. A single sample analysis was completed in 15 min, corresponding to approximately an order of magnitude improvement compared to a more conventional LC-MS approach.
2008. "Simultaneous Fragmentation of Multiple Ions Using IMS Drift Time Dependent Collision Energies." Journal of the American Society for Mass Spectrometry 19(3):411-419. doi:10.1016/j.jasms.2007.11.018 Abstract Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) was utilized to evaluate an ion collision energy ramping technique for simultaneously fragmenting all ions in a mixture without causing over- or under-fragmentation. To evaluate this technique, the fragmentation patterns of a mixture of ions ranging in mass, charge state and drift time were analyzed to determine their optimal fragmentation conditions. The precursor ions were pulsed into the IMS-MS instrument and separated in the IMS drift cell based on mobility differences. Two differentially pumped short quadrupoles were used to focus the ions exiting the IMS drift cell, and fragmentation was induced by collision induced dissociation (CID) between the conductance limiting orifice behind the second short quadrupole and before the first MS octopole. To explore the fragmentation spectrum of each precursor ion, the bias voltages for the short quadrupoles and the conductance limiting orifices were all increased from 0 V to 50 V above the non-fragmentation voltage settings. A linear correlation was observed between the optimal fragmentation voltage for each ion and their specific drift time, so a linear voltage gradient was employed to supply less collision energy to high mobility ions (e.g., small conformations or higher charge states) and more to low mobility ions. Fragmentation efficiencies were calculated for all ions, and similar values were observed when the fragmentation voltage was linearly ramped with IMS drift time, but varied drastically when only a single fragmentation voltage was used.
2008. "Proteomics Analysis of the Causative Agent of Typhoid Fever." Journal of Proteome Research 7(2):546-557. doi:10.1021/pr070434u Abstract Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. typhi). S. typhi infection is a complex process that involves numerous bacterially-encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study we used a liquid chromatography-mass spectrometry (LC-MS) based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins. In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions (Adkins J.N. et al (2006) Mol Cell Prot). Comparative proteomic analysis of S. typhi (strain Ty2) and S. typhimurium (strains LT2 and 14028) revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and a conserved protein encoded by t1476. The differential expression of selected proteins was confirmed by Western blot analysis. Taken together with the current literature, our observations suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity. In addition, we observed products of the biotin (bio) operon displayed a higher abundance in the more virulent strains S. typhi-Ty2 and S. typhimurium-14028 compared to the virulence attenuated S. typhimurium strain LT2, suggesting bio proteins may contribute to Salmonella pathogenesis.
2008. "Proteogenomics: the needs and roles to be filled by proteomics in genome annotation." Briefings in Functional Genomics and Proteomics 7(1):50-62. Abstract While genome sequencing efforts reveal the basic building blocks of life, a genome sequence alone is insufficient for elucidating biological function. Genome annotation – the process of identifying genes and assigning function to each gene in a genome sequence – provides the means to elucidate biological function from sequence. Current state-of-the-art high throughput genome annotation uses a combination of comparative (sequence similarity data) and non-comparative (ab initio gene prediction algorithms) methods to identify open reading frames in genome sequences. Because approaches used to validate the presence of these open reading frames are typically based on the information derived from the annotated genomes, they cannot independently and unequivocally determine whether a predicted open reading frame is translated into a protein. With the ability to directly measure peptides arising from expressed proteins, high throughput liquid chromatography-tandem mass spectrometry-based proteomics, approaches can be used to verify coding regions of a genomic sequence. Here, we highlight several ways in which high throughput tandem mass spectrometry-based proteomics can improve the quality of genome annotations and suggest that it could be efficiently applied during the initial gene calling process so that the improvements are propagated through the subsequent functional annotation process.
2007. "Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry." Journal of Proteome Research 6(6):2323-2330. doi:doi:10.1021/pr070112q Abstract Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.
2007. "Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides." Rapid Communications in Mass Spectrometry 21(5):661-666. doi:10.1002/rcm.2884 Abstract Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in diabetes mellitus research, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins.
2007. "A Method for Selective Enrichment and Analysis of Nitrotyrosine-Containing Peptides in Complex Proteome Samples." Journal of Proteome Research 6(6):2257-2268. doi:10.1021/pr0606934 Abstract Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and aging related pathologies; however, the lack of an efficient enrichment method has prevented the analysis of this important low level protein modification. We have developed an efficient method for specific enrichment of nitrotyrosine containing peptides that permits nitrotyrosine peptides and specific nitration sites to be unambiguously identified with LC-MS/MS. The method is based on the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process starts with acetylation with acetic anhydride to block all primary amines, followed by reduction of nitrotyrosine to aminotyrosine, then derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and finally deprotecting of S-acetyl on SATA to form free sulfhydryl groups. This method was evaluated using nitrotyrosine containing peptides, in-vitro nitrated human histone 1.2, and bovine serum albumin (BSA). 91% and 62% of the identified peptides from enriched histone and BSA samples were nitrotyrosine derivatized peptides, respectively, suggesting relative high specificity of the enrichment method. The application of this method to in-vitro nitrated mouse brain homogenate resulted in 35% of identified peptides containing nitrotyrosine (compared to only 5.9% observed from the global analysis of unenriched sample), and a total of 150 unique nitrated peptides covering 102 proteins were identified with a false discovery rate estimated at 3.3% from duplicate LC-MS/MS analyses of a single enriched sample.
2007. "Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition." Journal of Proteome Research 6(11):4489-4497. doi:10.1021/pr070068e Abstract To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.
2007. " Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus." Journal of Cell Science 120(22):4060-70. doi:10.1242/jcs.014795 Abstract The chromosomal passenger complex (CPC) is a critical regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation specific antibody that labels the CPC using liquid chromatography coupled to mass spectrometry. A mitotic phosphorylation motif (PX{G/T/S}{L/M}[pS]P or WGL[pS]P) was identified in 11 proteins including Fzr/Cdh1 and RIC-8, two proteins with potential links to the CPC. Phosphoprotein complexes contained known CPC components INCENP, Aurora-B and TD-60, as well as SMAD2, 14-3-3 proteins, PP2A, and Cdk1, a likely kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins including SMAD2, Plk3 and INCENP. Mitotic SMAD2 and Plk3 phosphorylation was confirmed using phosphorylation specific antibodies, and in the case of Plk3, phosphorylation correlates with its localization to the mitotic apparatus. A mutagenesis approach was used to show INCENP phosphorylation is required for midbody localization. These results provide evidence for a shared phosphorylation event that regulates localization of critical proteins during mitosis.
2007. "Head-to-Head Comparison of Serum Fractionation Techniques." Journal of Proteome Research 6(2):828-836. doi:10.1021/pr0604920 Abstract Multiple approaches for simplifying the serum proteome have been described. These techniques are generally developed across different laboratories, samples, mass spectrometry platforms, and analysis tools. Hence, comparing the available schemes is impossible from the existing literature because of confounding variables. We describe a head-to-head comparison of several serum fractionation schemes, including N-linked glycopeptide enrichment, cysteinyl-peptide enrichment, magnetic bead separation (C3, C8, and WCX), size fractionation, Protein A/G depletion, and immunoaffinity column depletion of abundant serum proteins. Each technique was compared to results obtained from unfractionated human serum. The results show immunoaffinity subtraction is the most effective means for simplifying the serum proteome and maintaining reasonable sample throughput. The reported dataset is publicly available and provides a standard against which emergent technologies can be compared and evaluated for their contribution to serum-based biomarker discovery.
2007. "Profiling Signaling Polarity in Chemotactic Cells." Proceedings of the National Academy of Sciences of the United States of America 104(20):8328-8333. doi:10.1073/pnas.0701103104 Abstract While directional movement requires morphological polarization characterized by formation of a leading pseudopodium at the front and a trailing rear at the back, little is known about how protein networks are spatially integrated to regulate this process. Here, we utilize a unique pseudopodial purification system and quantitative proteomics and phosphoproteomics to map the spatial relationship of 3509 proteins and 228 distinct sites of phosphorylation in polarized cells. Networks of signaling proteins, metabolic pathways, actin regulatory proteins, and kinase-substrate cascades were found to partition to different poles of the cell including components of the Ras/ERK pathway. Also, several novel proteins were found to be differentially phosphorylated at the front or rear of polarized cells and to localize to distinct subcellular structures. Our findings provide insight into the spatial organization of signaling networks that control cell movement and provide a comprehensive profile of proteins and their sites of phosphorylation that control cell polarization.
2007. "Methods for Pseudopodia Purification and Proteomic Analysis." Sciences STKE Signal Transduction Knowledge Environment 2007(400):p14. doi:DOI: 10.1126/stke.4002007pl4 Abstract Directional cell migration (chemotaxis) plays a central role in a wide spectrum of physiological and pathological processes, including embryo development, wounding healing, immunity, and cancer metastasis (1, 2). The process of chemotaxis is characterized by the sustained migration of cells in the direction of an increasing concentration of chemoattractant and/or ECM protein. Upon sensing the chemoattractant cells response with localized amplification of signals on the side facing the gradient (3-7). The spatial signal propagation facilitates reorganization of the actin-myosin cytoskeleton leading to extension of a dominant pseudopodium (PD) only in the direction of chemoattractant (7-10). While it is clear that localized signaling is critical for pseudopodium formation and chemotaxis, the molecular mechanisms that mediate this response remain poorly defined. To investigate mechanisms of pseudopodia formation, we recently described a novel approach to separate the PD and cell body (CB) compartments for large scale proteomic and phosphoproteomic analyses using chambers equipped with microporous filters (Fig. 1) (3, 7, 11). This in vitro system recapitulates physiological events associates with pseudopodial protrusion through small openings in the ECM and the vessel wall during immune cell intravasation and cancer cell metastasis (12, 13). The model system has been used to reveal important signaling pathways and novel proteins that mediate cell migration. This model, combined with the state-of-the-art proteomics and phosphoproteomics technology, will provide an effective approach to systematically analyze the proteins that differentially localized or phosphorylated in the front and the back of polarized migrating cells. In the following sections, we will describe in detail the protocols used to purify the PD and CB compartments for large-scale proteomic and phosphoproteomic analyses using mass spectrometry.
2007. "Identification of a Denitrase Activity Against Calmodulin in Activated Macrophages Using High-Field Liquid Chromatography - FTICR Mass Spectrometry." Biochemistry 46(37):10498-10505. Abstract We have identified a denitrase activity in macrophages that is upregulated following macrophage activation, which is shown by mass spectrometry to recognize nitrotyrosines in the calcium signaling protein calmodulin (CaM) and convert them to their native tyrosine structure without the formation of any aminotyrosine. Comparable extents of methionine sulfoxide reduction are also observed that are catalyzed by endogenous methionine sulfoxide reductases. Competing with repair processes, oxidized CaM is a substrate for a peptidase activity that results in the selective cleavage of the C-terminus lysine (i.e., Lys148) that is expected to diminish CaM function. Thus, competing repair and peptidase activities define the abundances and functionality of CaM to modulate cellular metabolism in response to oxidative stress, where the presence of the truncated CaM species provides a useful biomarker for the transient appearance of oxidized CaM.
2007. "Scaling of the Resolving Power and Sensitivity for Planar FAIMS and Mobility-Based Discrimination in Flow- and Field- Driven Analyzers." Journal of the American Society for Mass Spectrometry 18(9):1672–1681. doi:10.1016/j.jasms.2007.06.013 Abstract Continuing rapid development of the technology and applications of field asymmetric waveform ion mobility spectrometry (FAIMS) calls for better understanding of the limitations of this method and factors that govern them. While key performance metrics such as resolution and ion transmission efficiency had been calculated for specific cases using numerical simulations, the underlying physical trends remained obscure. Here we determine that the resolving power of planar FAIMS scales as the square root of separation time and sensitivity drops exponentially at the rate controlled by absolute ion mobility and several instrumental parameters. A strong dependence of ion transmission on mobility causes severe discrimination against species with higher mobility, presenting particular problems for analyses of complex mixtures. While the time evolution of resolution and sensitivity is virtually identical in existing FAIMS systems driven by gas flow and proposed devices driven by electric field, the distributions of separation times are not. The inverse correlation between mobility (and thus diffusion speed) and residence time for a particular species in field-driven FAIMS greatly reduces the mobility-based discrimination and provides much more uniform separations.
2007. "Optimization of Algorithms for Ion Mobility Calculations." Journal of Physical Chemistry A 111(10):2002-2010. doi:10.1021/jp066953m Abstract Ion mobility spectrometry (IMS) is increasingly employed to probe the structures of gas-phase ions, particularly those of proteins and other biological macromolecules. This process involves comparing measured mobilities with those computed for potential geometries, which requires evaluation of orientationally averaged cross sections using some approximate treatment of ion-buffer gas collisions. Two common models are the Projection Approximation (PA) and Exact Hard-Spheres Scattering (EHSS) that represent ions as collections of hard spheres. Though calculations for large ions and/or conformer ensembles take significant time, no algorithmic optimization had been explored. Previous EHSS programs were dominated by ion rotation operations that allow orientational averaging. We have developed two new algorithms for PA and EHSS calculations: one simplifies those operations and greatly reduces their number, and the other disposes of them altogether by propagating trajectories from a random origin. The new algorithms were tested for a representative set of seven ion geometries including diverse sizes and shapes. While the best choice depends on the geometry in a non-obvious way, the difference between the two codes is generally modest. Both are much more efficient than the existing software, for example faster than the widely used Mobcal (implementing EHSS) ~10 - 30 fold.
2007. "Distortion of Ion Structures by Field Asymmetric Waveform Ion Mobility Spectrometry." Analytical Chemistry 79(4):1523-1528. doi:10.1021/ac061306c Abstract Field asymmetric waveform ion mobility spectrometry (FAIMS) is emerging as a major analytical tool, especially in conjunction with mass spectrometry (MS) and/or conventional ion mobility spectrometry (IMS). In particular, FAIMS is used to separate protein or peptide conformers prior to characterization by IMS, MS/MS, or H/D exchange. High electric fields in FAIMS induce ion heating, previously estimated at <10 0C on average and believed too weak to affect ion geometries. Here we use a FAIMS/IMS/MS system to compare the IMS spectra for ESI-generated ubiquitin ions that have and have not passed FAIMS, and find that some unfolding occurs for all charge states. The analysis of those data and their comparison with reported protein unfolding in a Paul trap indicate that at least some structural transitions observed in FAIMS, or previously in an ion trap, are not spontaneous. The observed unfolding is overall similar to that produced by heating of ~40 - 50 0C above room temperature, consistent with the calculated heating of ions at FAIMS waveform peaks. Hence the isomerization in FAIMS likely proceeds in steps during “hot” periods, especially right after ions entering the device. That process distorts ion geometries and causes ion losses by a “self-cleaning” mechanism, and thus should be suppressed as much as possible. We propose achieving that via cooling FAIMS by the amount of ion heating; in most relevant cases cooling by ~75 0C should suffice.
2007. "Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry." Journal of Proteome Research 6(2):602-610. doi:10.1021/pr060354a Abstract We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.
2007. "Spatial Mapping of Protein Abundances in the Mouse Brain by Voxelation Integrated with High-Throughput Liquid Chromatography ─ Mass Spectrometry." Genome Research 17(3):328-336. doi:doi:10.1101/gr.5799207 Abstract Temporally and spatially resolved mapping of protein abundance patterns within the mammalian brain is of significant interest for understanding brain function and molecular etiologies of neurodegenerative diseases; however, such imaging efforts have been greatly challenged by complexity of the proteome, throughput and sensitivity of applied analytical methodologies, and accurate quantitation of protein abundances across the brain. Here, we describe a methodology for comprehensive spatial proteome mapping that addresses these challenges by employing voxelation integrated with automated microscale sample processing, high-throughput LC system coupled with high resolution Fourier transform ion cyclotron mass spectrometer and a “universal” stable isotope labeled reference sample approach for robust quantitation. We applied this methodology as a proof-of-concept trial for the analysis of protein distribution within a single coronal slice of a C57BL/6J mouse brain. For relative quantitation of the protein abundances across the slice, an 18O-isotopically labeled reference sample, derived from a whole control coronal slice from another mouse, was spiked into each voxel sample and stable isotopic intensity ratios were used to obtain measures of relative protein abundances. In total, we generated maps of protein abundance patterns for 1,028 proteins. The significant agreement of the protein distributions with previously reported data supports the validity of this methodology, which opens new opportunities for studying the spatial brain proteome and its dynamics during the course of disease progression and other important biological and associated health aspects in a discovery-driven fashion.
2007. "Ionization and transmission efficiency in an electrospray ionization-mass spectrometry interface." Journal of the American Society for Mass Spectrometry 18(9):1582-1590. doi:10.1016/j.jasms.2007.05.018 Abstract The efficiency of sample ionization by electrospray ionization (ESI) and the transmission of the charged droplets and gas-phase ions through an ESI interface were investigated in order to advance the understanding of how these factors affect mass spectrometry (MS) sensitivity. In addition, the effects of the ES emitter distance to the inlet, solution flow rate, and inlet temperature to the ionization and transmission efficiency were characterized. Quantitative measurements of ES current loss throughout the ESI interface were accomplished by electrically isolating the front surface of the interface from the inner wall of the heated inlet capillary, enabling losses on the two surfaces to be distinguished. The ES current lost to the front surface of the ESI interface was also spatially profiled with a linear array of 340-µm-dia. electrodes placed adjacent to the inlet capillary entrance. Current transmitted as gas-phase ions was differentiated from charged droplets and solvent clusters by directly measuring sensitivity with a single quadrupole mass spectrometer. The study has revealed a large sampling efficiency into the inlet capillary (>90% at an emitter distance of 1 mm), a global rather than a local gas dynamic effect on the shape of the ES plume due to the gas flow conductance limit of the inlet capillary, a large (>80%) loss of analyte after transmission through the inlet due to incomplete desolvation at a solution flow rate of 1.0 µL/min, and a decrease in analyte peak intensity at lower temperatures, despite a large increase in ES current transmission efficiency. These studies provide a clearer understanding of the parameters affecting ion transmission into the mass spectrometer, and will serve to guide the design of more efficient instrument interfaces.
2007. "An electrodynamic ion funnel interface for greater sensitivity and higher throughput with linear ion trap mass spectrometers." International Journal of Mass Spectrometry 265(2-3):244-250. doi:10.1016/j.ijms.2007.02.032 Abstract An electrospray ionization interface incorporating an electrodynamic ion funnel has been designed and implemented in conjunction with a linear ion trap mass spectrometer (Thermo Electron, LTQ). We found ion transmission to be greatly improved by replacing the standard capillary-skimmer interface with the capillary-ion funnel interface. An infusion study using a serial dilution of a reserpine solution showed that ion injection times to fill the ion trap were reduced by ~90% which resulted in an ~10-fold increase in reported peak intensities. In liquid chromatography (LC)-MS and LC tandem MS (MS/MS) experiments performed using a proteomic sample from the bacterium, Shewanella oneidensis, the ion funnel interface provided an ~7-fold reduction in ion injection (accumulation) times. In a series of LC-MS/MS experiments we found that more dilute S. oneidensis samples provided more peptide and protein identifications when the ion funnel interface was used in place of the standard interface. This improvement was most pronounced at lower sample concentrations, where extended ion accumulation times are required, resulting in an ~2-fold increase in the number of protein identifications. Implementation of the ion funnel interface with a LTQ Fourier transform (FT) MS requiring much greater ion populations resulted in spectrum acquisition times reduced by ~25 to 50%.
2007. "VIPER: an advanced software package to support high-throughput LC-MS peptide identification." Bioinformatics 23(15- August 2007):2021-2023. doi:10.1093/bioinformatics/btm281 Abstract High throughput liquid chromatograph-mass spectrometry (LC-MS) based proteomics analyses have necessitated development of software to manipulate large volumes of detailed data and produce confident peptide/protein identifications. VIPER unites important data processing steps in a single software package that can be used to visualize peptide mass and LC elution (i.e. retention) time “feature” relationships from individual analyses, match these LC-MS features to accurate mass and time (AMT) tags of peptides previously identified in LC-MS/MS analyses, and to identify and quantify pairs of isotopically labeled peptides.
2007. "The future of liquid chromatography-mass spectrometry in metabolic profiling and metabolomic studies for biomarker discovery." Biomarkers in Medicine 1(1):159-185. doi:doi: 10.2217/17520363.1.1.159 Abstract The future utility of liquid chromatography-mass spectrometry (LC-MS) in metabolic profiling and metabolomic studies for biomarker discover will be discussed, beginning with a brief description of the evolution of metabolomics and the utilization of the three most popular analytical platforms in such studies: NMR, GC-MS, and LC-MS. Emphasis is placed on recent developments in high-efficiency LC separations and sensitive electrospray ionization approaches and the benefits to incorporating both in LC-MS-based approaches. The advantages and disadvantages of various quantitative approaches are reviewed, followed by the current LC-MS-based tools available for candidate biomarker characterization and identification. Finally, a brief prediction on the future path of LC-MS-based methods in metabolic profiling and metabolomic studies is given.
2007. "Electrospray characteristic curves: in pursuit of improved performance in the nano-flow regime." Analytical Chemistry 79(21):8030-8036. doi:10.1021/ac070800t Abstract Depending on its coordinates in the parameter space, an electrospray can manifest in one of several known regimes – stable, quasi-stable, transitional-chaotic, and non-axial – that ultimately impact measurement sensitivity and precision. An electrospray operating in cone-jet regime provides large and stable spray current, as well as smaller initial droplets that are prerequisites for higher sensitivity and quality mass spectrometric analyses. However, the dynamic conditions encountered in gradient elution-based liquid separations create difficulties for continuous operation in this regime throughout the analysis. We present a preliminary study aimed at stabilizing the electrospray in the cone-jet regime. On the basis of spray current measurements obtained using solvent conditions typically found in liquid chromatography-mass spectrometry, an improved description of the cone-jet stability island is provided by including transitions to and from the recently described astable regime. Additionally, the experimental conditions in which the astable regime marks the transition between pulsating and cone-jet regimes are further clarified.
2007. "Targeted Protein Degradation by Salmonella under Phagosome-Mimicking Culture Conditions Investigated Using Comparative Peptidomics." Molecular & Cellular Proteomics. MCP 6(4):717-727. doi:10.1074/mcp.M600282-MCP200 Abstract The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Due to the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g., contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study Salmonella enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal compartment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified 5,163 distinct peptides originating from 682 proteins and the data clearly indicated that compared to cells cultured in the rich medium, Salmonella cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed by using bottom-up proteomics, and when the peptidomic and proteomic data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.
2007. "MicroSPE-nanoLC-ESI-MS/MS using 10-µm-i.d. silica-based monolithic columns for proteomics." Analytical Chemistry 79(2):540-545. Abstract Silica-based monolithic narrow bore capillary columns (25 cm 10 µm i.d.) with an integrated ESI emitter has been developed to provide high quality and robust microSPE-nanoLC-ESI-MS analyses. The integrated ESI emitter adds no dead volume to the LC separation, allowing stable electrospray performance to be obtained at flow rates of 10 nL/min. In an initial application we identified 5510 unique peptides covering 1443 distinct Shewanella oneidensis proteins from a 300 ng tryptic digest sample in a single 4-h LC-MS/MS analysis using a linear ion trap MS (LTQ). We found the use of an integrated monolithic ESI emitter provided enhanced resistance to clogging and good run-to-run reproducibility, with an average variation of ~25% for triplicate analyses.
2007. "Accurate Mass Measurements in Proteomics." Chemical Reviews 107(8):3621-3653. doi:10.1021/cr068288j Abstract To understand different aspects of life at the molecular level, one would think that ideally all components of specific processes should be individually isolated and studied in details. Reductionist approaches, i.e., studying one biological event at a one-gene or one-protein-at-a-time basis, indeed have made significant contributions to our understanding of many basic facts of biology. However, these individual “building blocks” can not be visualized as a comprehensive “model” of the life of cells, tissues, and organisms, without using more integrative approaches.1,2 For example, the emerging field of “systems biology” aims to quantify all of the components of a biological system to assess their interactions and to integrate diverse types of information obtainable from this system into models that could explain and predict behaviors.3-6 Recent breakthroughs in genomics, proteomics, and bioinformatics are making this daunting task a reality.7-14 Proteomics, the systematic study of the entire complement of proteins expressed by an organism, tissue, or cell under a specific set of conditions at a specific time (i.e., the proteome), has become an essential enabling component of systems biology. While the genome of an organism may be considered static over short timescales, the expression of that genome as the actual gene products (i.e., mRNAs and proteins) is a dynamic event that is constantly changing due to the influence of environmental and physiological conditions. Exclusive monitoring of the transcriptomes can be carried out using high-throughput cDNA microarray analysis,15-17 however the measured mRNA levels do not necessarily correlate strongly with the corresponding abundances of proteins,18-20 The actual amount of functional proteins can be altered significantly and become independent of mRNA levels as a result of post-translational modifications (PTMs),21 alternative splicing,22,23 and protein turnover.24,25 Moreover, the functions of expressed proteins can also be extensively modified by PTMs26-31 or by their interactions with other biomolecules or small molecules.32,33 Thus, it is highly desirable that proteins, the primary functional macromolecules involved in almost all biological activities, can be studied directly and systematically to determine their diverse properties and interplay. Such proteome-wide analysis is expected to provide a wealth of biological information, such as sequence, quantity, PTMs, interactions, activities, subcellular distribution and structure of proteins, which is critical to the comprehensive understanding of the biological systems. However, the de novo analysis of proteins isolated from cells, tissues or bodily fluids poses significant challenges due to the tremendous complexity and depth of the proteome, which necessitates high-throughput and highly sensitive analytical techniques. It is therefore not surprising that mass spectrometry (MS) has become an indispensable technology for proteome analysis.
2007. "Phosphopeptide elution times in reversed-phase liquid chromatography." Journal of Chromatography A 1172(1):9-18. doi:doi:10.1016/j.chroma.2007.09.032 Abstract Elution time shifts between 33 different peptides and their corresponding phosphopeptides ranging from 4 amino acid residues to 35 amino acids in length were systematically investigated utilizing a high resolution reversed-phase liquid chromatography (RPLC) system. Observed peptide elution time shifts for a single phosphorylation ranged from -5.28 min (for pYVPML) to +0.59 min (for HRDpSGLLDSLGR). Peptides containing a phosphotyrosine residue displayed a significant decrease in elution time following phosphorylation compared to their similar-sized peptides with phosphoserine or phosphothreonine residues. While the observed elution time generally decreased due to phosphorylation, five peptides displayed increased elution time as a result of phosphorylation. For large peptides (≥ 18 amino acids), the elution time shifts due to single phosphorylation were limited (ranging between -0.48 min and +0.03 min), while the elution time shifts for small peptides (< 18 amino acids) were characterized by a larger deviation (ranging between -5.28 min and +0.59 min). The predictive capability for the observed RPLC elution time change due to phosphorylation has been suggested, which will aid in assigning confident phosphopeptide identifications and their subsequent confirmation.
2007. "Array of Chemically Etched Fused Silica Emitters for Improving the Sensitivity and Quantitation of Electrospray Ionization Mass Spectrometry." Analytical Chemistry 79(11):4192-4198. doi:10.1021/ac062417e Abstract An array of emitters has been developed for increasing the sensitivity of electrospray ionization mass spectrometry (ESI-MS). The linear array consists of 19 chemically etched fused silica capillaries arranged with 500 µm (center-to-center) spacing. The multi-emitter device has a low dead volume to facilitate coupling to capillary liquid chromatography (LC) separations. The high aspect ratio of the emitters enables operation at flow rates as low as 20 nL/min/emitter, effectively extending the benefits of nanoelectrospray to higher flow rate analyses. To accommodate the larger ion current produced by the emitter array, a multi-capillary inlet to the mass spectrometer was also constructed. The inlet, which matched the dimensions of the emitter array, effectively preserved ion transmission efficiency. Standard reserpine solutions of varying concentration were electrosprayed at 1 µL/min using the multi-emitter/multi-inlet combination, and compared to a standard, single emitter configuration. A nine-fold sensitivity enhancement was observed for the multi-emitter relative to the single emitter. A bovine serum albumin tryptic digest was also analyzed and resulted in a sensitivity increase ranging from 2.4 to 12.3-fold for the detected tryptic peptides; the varying response was attributed to reduced ion suppression under the nano-ESI conditions afforded by the emitter array. An equimolar mixture of leucine enkephalin and maltopentaose was studied to verify that ion suppression is indeed reduced for the multi-ESI array relative to a single emitter over a range of flow rates.
2007. "Targeted Tandem Mass Spectrometry for High-Throughput Comparative Proteomics Employing NanoLC-FTICR MS with External Ion Dissociation." Journal of the American Society for Mass Spectrometry 18(7):1332-1343. doi:doi:10.1016/j.jasms.2007.04.011 Abstract ABSTRACT-Targeted tandem mass spectrometry (MS/MS) is an attractive proteomic approach that allows selective identification of peptides exhibiting abundance differences between culture conditions and/or diseased states. Herein, we report on a targeted LC-MS/MS capability realized with a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a quadrupole interface that provides data-dependent ion selection, accumulation, and dissociation externally to the ICR trap. Identification of a subset of differentially abundant proteins from Shewanella oneidensis grown under suboxic vs. aerobic conditions demonstrates the feasibility of such approach. High mass resolution offered by FTICR and effective on-the-fly elution time correction facilitated accurate selection of targets, while high mass measurement accuracy MS/MS data resulted in unambiguous peptide identifications.
2007. "Ion Funnel Trap Interface for Orthogonal Time-of-Flight Mass Spectrometry ." Analytical Chemistry 79(20):7845-7852. doi:10.1021/ac071091m Abstract A combined electrodynamic ion funnel and ion trap coupled to an orthogonal acceleration (oa)-time-of-flight mass spectrometer was developed and characterized. The ion trap was incorporated through the use of added terminal electrodynamic ion funnel electrodes enabling control over the axial dc gradient in the trap section. The ion trap operates efficiently at a pressure of ~1 Torr, and measurements indicate a maximum charge capacity of ~3 × 107 charges. An order of magnitude increase in sensitivity was observed in the analysis of low concentration peptides mixtures with orthogonal acceleration (oa)-time-of-flight mass spectrometry (oa-TOF MS) in the trapping mode as compared to the continuous regime. A signal increase in the trapping mode was accompanied by reduction in the chemical background, due to more efficient desolvation of, for example, solvent related clusters. Controlling the ion trap ejection time was found to result in efficient removal of singly charged species and improving signal-to-noise ratio (S/N) for the multiply charged analytes.
2007. "Whole proteome analysis of post-translational modifications: applications of mass-spectrometry for proteogenomic annotation." Genome Research 17(9):1362-1377. doi:0.1101/gr.6427907 Abstract While bacterial genome annotations have significantly improved in recent years, techniques for bacterial proteome annotation (including post-translational chemical modifications, signal peptides, proteolytic events, etc.) are still in their infancy. At the same time, the number of sequenced bacterial genomes is rising sharply, far outpacing our ability to validate the predicted genes, let alone annotate bacterial proteomes. In this study, we use tandem mass spectrometry (MS/MS) to annotate the proteome of Shewanella oneidensis MR-1, an important microbe for bioremediation. In particular, we provide the first comprehensive map of post-translational modifications in a bacterial genome, including a large number of chemical modifications, signal peptide cleavages and cleavage of N-terminal methionine residues. We also detect multiple genes that were missed or assigned incorrect start positions by gene prediction programs and suggest corrections to improve the gene annotation. This study demonstrates that complementing every genome sequencing project by an MS/MS project would significantly improve both genome and proteome annotations for a reasonable cost.
2007. "Quantitative Proteomic Approaches for Studying Phosphotyrosine Signaling." Expert Review of Proteomics 4(1):13-23. doi:doi:10.1586/14789450.4.1.13 Abstract Protein tyrosine phosphorylation is a fundamental mechanism for controlling many aspects of cellular processes, as well as aspects of human health and diseases. Compared to phosphoserine (pSer) and phosphothreonine (pThr), phosphotyrosine (pTyr) signaling is more tightly regulated, but often more challenging to characterize due to significantly lower level of tyrosine phosphorylation (a relative abundance of 1800:200:1 was estimated for pSer/pThr/pTyr in vertebrate cells[1]). In this review, we outline the recent advances in analytical methodologies for enrichment, identification, and accurate quantitation of tyrosine phosphorylated proteins and peptides using antibody-based technologies, capillary liquid chromatography (LC) coupled with mass spectrometry (MS), and various stable isotope labeling strategies, as well as non-MS-based methods such as protein or peptide array methods. These proteomic technological advances provide powerful tools for potentially understanding signal transduction at the system level and provide a basis for discovering novel drug targets for human diseases. [1] Hunter, T. (1998) The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 353, 583–605
2007. "Capillary LC Coupled with High-Mass Measurement Accuracy Mass Spectrometry for Metabolic Profiling." Analytical Chemistry 79(16):6081-6093. doi:10.1021/ac070064t Abstract We have developed an efficient and robust high-pressure capillary LC-MS method for the identification of large numbers of metabolites in biological samples using both positive and negative ESI modes. Initial efforts focused on optimizing the separations conditions for metabolite extracts using various LC stationary phases in conjunction with multiple mobile phase systems, as applied to the separation of 45 metabolite standards. The optimal mobile and stationary phases of those tested were determined experimentally (in terms of peak shapes, theoretical plates, retention of small, polar compounds, etc.), and both linear and exponential gradients were applied in the study of metabolite extracts from the cyanobacterium Cyanothece sp. ATCC 51142. Finally, an automated dual-capillary LC system was constructed and evaluated for the effectiveness and reproducibility of the chromatographic separations using the above samples. When coupled with a commercial LTQ-Orbitrap MS, ~900 features were reproducibly detected from Cyanothece sp. ATCC 51142 metabolite extracts. In addition, 12 compounds were tentatively identified, based on accurate mass, isotopic distribution, and MS/MS information.
2007. "Proteomic Profiling of Human Liver Biopsies: Hepatitis C Virus-Induced Fibrosis and Mitochondrial Dysfunction." Hepatology 46(3):649-657. doi:10.1002/hep.21751 Abstract Liver biopsies from HCV-infected patients offer the unique opportunity to study human liver biology and disease in vivo. However, the low protein yields associated with these small samples present a significant challenge for proteomic analysis. In this study we describe the application of an ultra-sensitive proteomics platform for performing robust quantitative proteomic studies on microgram amounts of HCV-infected human liver tissue from 15 patients at different stages of fibrosis. A high quality liver protein data base containing 5,920 unique protein identifications supported high throughput quantitative studies using 16O:18O stable isotope labeling in combination with the accurate mass and time (AMT) tag approach. A total of 1,641 liver biopsy proteins were quantified and ANOVA identified 210 proteins exhibiting statistically significant differences associated with fibrosis stage. Hierarchical clustering revealed that biopsies representative of later fibrosis stages (e.g. Batts-Ludwig stages 3-4) exhibited a distinct protein expression profile indicating an apparent down-regulation of many proteins when compared to samples from earlier fibrosis stages (e.g. Batts-Ludwig stages 0-2). Functional analysis of these signature proteins suggests that impairment of key mitochondrial processes including fatty acid oxidation and oxidative phosphorylation, and response to oxidative stress and reactive oxygen species occurs during advanced stage 3-4 fibrosis. In conclusion, the results reported here represent a significant advancement in clinical proteomics providing to our knowledge, the first demonstration of global proteomic alterations accompanying liver disease progression in patients chronically infected with HCV. Our findings contribute to a generally emerging theme associating oxidative stress and hepatic mitochondrial dysfunction with HCV pathogenesis.
2007. "A genome-scale map of expression for a mouse brain section obtained using voxelation." Physiological Genomics 30(3):313-321. Abstract Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological diseases. We have reconstructed 2- dimensional images of gene expression for 20,000 genes in a coronal slice of the mouse brain at the level of the striatum by using microarrays in combination with voxelation at a resolution of 1 mm3. Good reliability of the microarray results were confirmed using multiple replicates, subsequent quantitative RT-PCR voxelation, mass spectrometry voxelation and publicly available in situ hybridization data. Known and novel genes were identified with expression patterns localized to defined substructures within the brain. In addition, genes with unexpected patterns were identified and cluster analysis identified a set of genes with a gradient of dorsal/ventral expression not restricted to known anatomical boundaries. The genome-scale maps of gene expression obtained using voxelation will be a valuable tool for the neuroscience community.
2007. "Quantitative analysis of human immunodeficiency virus type 1-infected CD4(+) cell proteome: Dysregulated cell cycle progression and nuclear transport coincide with robust virus production." Journal of Virology 81(14):7571-7583. Abstract Relatively little is known at the functional genomic level about the global host response to HIV-1 infection. Microarray analyses by several laboratories, including our own, have revealed that human immunodeficiency virus type 1 infection causes significant changes in host mRNA abundance and regulation of several cellular biological pathways. However, it remains unclear what consequences these changes bring about at the protein level. Here we report the expression levels of ~3,200 proteins assessed in the CD4+ CEMx174 cell line after infection with HIV-1 LAI, using liquid chromatography-mass spectrometry coupled with stable isotope labeling and the accurate mass and time (AMT) tag approach. Further, we found that 687 (21%) proteins changed in abundance at the peak of virus production at 36h post-infection. Pathway analysis revealed that the differential expression of proteins were concentrated in select biological pathways, exemplified by ubiquitin conjugating enzymes in the ubiquitination, carrier proteins in nucleo-cytoplasmic transport, cyclin-dependent kinase in cell cycle progression, and pyruvate dehydrogenase of the citrate cycle. Moreover, we observed changes in the abundance of proteins with known interactions with HIV-1 viral proteins. Our proteomic analysis captured changes in the host protein milieu at the time of robust virus production, accompanied by a moderate accumulation of G1/G0-phase cells. We will discuss the contributions of these changes to virus production in the infected cells.
2007. "Multiplexed Ion Mobility Spectrometry - Orthogonal Time-Of-Flight Mass Spectrometry ." Analytical Chemistry 79(6):2451-2462. doi:10.1021/ac0617316 Abstract Ion mobility spectrometry (IMS) coupled to orthogonal time-of-flight mass spectrometry (TOF) has shown significant promise for the characterization of complex biological mixtures. The enormous complexity of biological samples (e.g. from proteomics) and the need for both biological and technical analysis replicates imposes major challenges for multidimensional separation platforms in regard to both sensitivity and sample throughput. A major potential attraction of the IMS-TOF MS platform is separation speeds exceeding that of conventional condensed-phase separations by orders of magnitude. Known limitations of the IMS-TOF MS platforms that presently mitigate this attraction include the need for extensive signal averaging due to factors that include significant ion losses in the IMS-TOF interface and an ion utilization efficiency of less than ~1% with continuous ion sources (e.g. ESI). We have developed a new multiplexed ESI-IMS-TOF mass spectrometer that enables lossless ion transmission through the IMS-TOF as well as a utilization efficiency of >50% for ions from the ESI source. Initial results with a mixture of peptides show a ~10-fold increase in signal-to-noise ratio with the multiplexed approach compared to a signal averaging approach, with no reduction in either IMS or TOF MS resolution.
2007. "Ion Mobility Spectrometry – Mass Spectrometry Performance Using Electrodynamic Ion Funnels and Elevated Drift Gas Pressures." Journal of the American Society for Mass Spectrometry 18(7):1176-1187. doi:doi:10.1016/j.jasms.2007.03.031 Abstract The ability of ion mobility spectrometry coupled with mass spectrometry (IMS-MS) to characterize biological mixtures has been illustrated over the past eight years. However, the challenges posed by the extreme complexity of many biological samples have demonstrated the need for higher resolution IMS-MS measurements. We have developed a higher resolution ESI-IMS-TOF MS by utilizing high pressure electrodynamic ion funnels at both ends of the IMS drift cell and operating the drift cell at an elevated pressure compared to a previous design. The ESI-IMS-TOF MS instrument consists of an ESI source, an hourglass ion funnel used for ion accumulation/injection into an 88 cm drift cell followed by a 10 cm ion funnel and a commercial orthogonal time-of-flight mass spectrometer providing high mass measurement accuracy. It was found that the rear (exit) ion funnel could be effectively operated as an extension of the drift cell when the DC fields were matched, allowing the instrument to have an effective drift region of 98 cm. Two differentially pumped quadrupole regions were used to couple the IMS and TOF MS to focus and minimize the ion transient time between the stages. The resolution of the instrument was evaluated at pressures ranging from 4 to12 Torr and ion mobility drift voltages of 16 V/cm (4 Torr) to 43 V/cm (12 Torr). An increase in resolution from 55 to 80 was observed from 4 to 12 Torr nitrogen drift gas with no loss in sensitivity. Given the increased usage of ion funnels prior to ion mobility separations, additional attention was directed towards the influence of drift gas on the observed ion populations trapped and transmitted using an electrodynamic ion funnel. The choice of drift gas was shown to influence the degree of ion heating and relative trapping efficiency within the ion funnel.
2006. "A Proteomics Analysis of Human Cytomegalovirus Particles." Chapter 5 in Cytomegaloviruses: Molecular Biology and Immunology, ed. Matthias J. Reddehase, pp. 91-110. Caister Academic Press, Hethersett, United Kingdom. Abstract While the sequence of the AD169 HCMV genome has been known for several years, the viral and cellular proteins that compose the infectious HCMV virion and entry-competent, non-replicating viral particles such as Dense Bodies (DBs) and Non-Infectious Enveloped Particles (NIEPs) are unknown. To approach this problem we have utilized a gel-free 2-D capillary liquid chromatography (LC)-MS/MS and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to identify and determine the relative abundance of viral and cellular proteins in purified HCMV AD169 particles. !is study has identified and quantitated the proteins that compose both HCMV virions and DBs. While a number of previously identified proteins were detected by this method the number of viral proteins that compose the HCMV virion was doubled in this study suggesting that over a third of the viral open reading frames are part of an infectious virion. !is chapter will discuss the implications of our findings in relation to what was previously known about HCMV and MCMV virion composition.
2006. "AMT Tag Approach to Proteomic Characterization of Deinococcus Radiodurans and Shewanella Oneidensis ." In Microbial Proteomics, Methods of Biochemical Analysis, vol. 49, ed. I. Humphery-Smith and M. Hecker, pp. 113-134. John Wiley & Sons, Inc., Hoboken, NJ. Abstract Biology is transitioning from a largely qualitative, mostly descriptive science to a quantitative and ultimately predictive science. Advances in high throughput DNA sequencing have made increasing numbers of genome sequences available and enabled a “systems” level analysis of complex biological organisms. The ability to quantitatively measure the array of proteins, also termed the proteome, in prokaryotic cells and communities of cells is key to understanding microbial systems. This chapter focuses on the utility of the AMT tag mass spectrometric approach used to characterize the proteomes of two microbes, Deinococcus radiodurans and Shewanella oneidensis MR-1.
2006. "Proteomic Analyses using High-Efficiency Separations and Accurate Mass Measurements." Chapter 20 in Separations in Proteomics , ed. Smejkal, GB and A Lazareu, pp. 363-385. CRC Taylor and Francis Group, New York, NY. Abstract While the development of analytical techniques for detecting and identifying proteins has been an actively pursued area of research for many years, a recent and concerted reemergence of work in this area has resulted in an explosion of new developments that can be applied across multiple disciplines. The advent of numerous mass spectrometric and separation advances may be viewed as the technical driving force behind these developments, but other factors are perhaps equally crucial. These factors include the increasing availability of sequences for many organisms as a result of recent advancements in genome analysis, recognition that there are limitations to using sequence/transcriptome information alone, and the more apparent value of directly characterizing the biomolecules and pathways that actually drive cellular events. Direct identification of structural and functional proteins allows perturbations at the protein level to be linked to responses at the cellular level, and provides a method for analyzing global proteomic changes in virtually any model system. The field of proteomics, in particular, has benefited from numerous recent analytical advances that have provided increased sensitivity and dynamic range of proteins detected, as well as analysis throughput. Traditional approaches typically have involved separations using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) in conjunction with a mass spectrometric (MS) component for protein identification.[1] These seminal techniques set the foundation for the advancing field of proteomics. The limitations associated with the gel-based techniques are well known and stem mostly from a lack of sufficient dynamic range needed for in-depth proteome coverage and [2] and from the large amount of time needed for analysis, which dramatically limits throughput. Significant developments have been made in an attempt to address both these and other issues. With the advent of electrospray ionization, researchers can now couple multi-dimensional liquid chromatography (LC) separations with MS analysis. Combined with a “bottom up” strategy, in which proteins are digested into smaller peptide fragments for identification, the LC-MS platform has proved highly successful for identifying peptides (and generally proteins) from complex samples. This chapter discusses proteome analysis techniques that are based on high resolution LC separations and high mass accuracy MS instrumentation, specifically the use of Fourier transform ion cyclotron resonance MS (FTICR), and their application for high throughput global identification of proteins. The simultaneously expanded sensitivity, dynamic range, and mass accuracy afforded by FTICR place this system at the forefront of analytical systems that can attain proteomic identifications from complex biological samples. We will begin the discussions by describing a high throughput proteomics approach that capitalizes on the attributes of FTICR. The critically important supporting components that combine to provide a comprehensive proteomic analysis capability—electrospray ionization efficiency, pre-MS high efficiency LC separations, and downstream data processing methods—will then be discussed with the intent of providing insight into the process as a whole, as well as to highlight analytical challenges that remain to be addressed. Because quantitative measurements are so important to understanding even the simplest biological systems, some of the quantitation techniques and approaches being developed and applied in our laboratory are also discussed. Several applications are exemplified prior to concluding this chapter.
2006. "Advances in Proteomics Data Analysis and Display Using an Accurate Mass and Time Tag Approach ." Mass Spectrometry Reviews 25(3):450-482. Abstract Proteomics, and the larger field of systems biology, have recently demonstrated utility in both the understanding of cellular processes on the molecular level and the identification of potential biomarkers of various disease states. The large amount of data generated by utilizing high mass accuracy mass spectrometry for high-throughput proteomics analyses presents a challenge in data processing, analysis and display. This review focuses on recent advances in nanoLC-FTICR-MS-based proteomics analysis and the accompanying data processing tools that have been developed in order to interpret and display the large volumes of data produced.
2006. "LC-MS/MS Based Proteomic Analysis and Functional Inference of Hypothetical Proteins in Desulfovibrio Vulgaris." Biochemical and Biophysical Research Communications 349(4):1412-1419. doi:10.1016/j.bbrc.2006.09.019 Abstract Direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions (Zhang et al., 2006a, Proteomics, 6: 4286-4299), this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Across six growth conditions, 15,841 tryptic peptides were identified with high confidence. Using a criterion of peptide identification from at least two out of three independent LC-MS/MS analyses per protein, 176 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. These proteins ranged from 6.0 to 153 kDa, and had calculated pI values ranging from 3.7 to 11.5. Based on homology search results (with E value <= 0.01 as a cutoff), 159 proteins were defined as conserved hypothetical proteins, and 17 proteins were unique to the D. vulgaris genome. Functional inference of the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 27 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification for the authenticity of hypothetical proteins and improve annotation for the D. vulgaris genome.
2006. "A Proteomic View of Desulfovibrio Vulgaris Metabolim as Determined by Liquid Chromatography Coupled with Tandem Mass Spectrometry." Proteomics 6(15):4286-4299. doi:10.1002/pmic.200500930 Abstract Direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins from Desulfovibrio vulgaris grown at exponential or stationary phase on a minimal medium containing either lactate or formate as the primary carbon source, with the goal of an initial characterization of the diversity of proteins synthesized under these conditions. Across all four growth conditions, 976 gene products were identified with high confidence, which is equal to approximately 28% of all predicted proteins in the D. vulgaris genome. Among these, fifty-two out of 55 predicted ribosomal proteins (~95%) were identified with high confidence. Functional analysis showed that the proteins identified were distributed among almost all functional classes, with the energy metabolism category containing the greatest number of identified proteins. At least 154 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins, which provided verification for the authenticity of these hypothetical proteins. Proteomic analysis showed that members of the proton gradient pathway, catalyzed by alcohol dehydrogenases and heterodisulfide reductases, and [NiFe] hydrogenase (HynAB-1) of the hydrogen cycling pathway were highly expressed in all four growth conditions, suggesting they may be the primary pathways for ATP synthesis in D. vulgaris. Most of the enzymes involved in substrate-level phosphorylation were also detected in all tested conditions. However, no enzyme involved in CO cycling or formate cycling was detected, suggesting these are not the primary pathways for ATP biosynthesis under the tested conditions. This study provides the first proteomic overview of the cellular metabolism of D. vulgaris.
2006. "Phosphoproteome Profiling of Human Skin Fibroplast Cells in Response to Low- and High-Dose Irradiation ." Journal of Proteome Research 5(5):1252-1260. Abstract The biological effect of low-dose radiation is currently not well understood. A hallmark of the response to radiation is the phosphorylation of proteins involved in DNA repair, DNA damage signaling, and cell cycle checkpoint control, which is important in prompt cellular response. The objective of the work presented here was to explore the phosphoproteome of normal human skin fibroblast (HSF) cells to reveal differences between low- and high-dose irradiation responses at the protein phosphorylation level. Several techniques —Trizol extract of proteins, methylation of the enzyme digest (peptides), enrichment of phosphopeptides with immobilized metal affinity chromatography (IMAC), nanoflow reversed-phase HPLC (nano-LC)/electrospray ionization, and tandem mass spectrometry— were combined for analysis of the HSF cell phosphoproteome following low- and high-doses of irradiation. More than 95% of the peptides identified after IMAC enrichment were phosphopeptides. Among the 493 unique phosphopeptides, 232 were singly phosphorylated, 220 were doubly phosphorylated, and 41 were triply phosphorylated, indicating the overall effectiveness of the IMAC technique to enrich both singly and multiple phosphorylated peptides. Over 700 phosphorylation sites were assigned to a total of 346 proteins, many of which are known or proposed to be highly relevant to a plethora of fundamental biological processes. The profile for proteins identified from the low-dose (2cGy) irradiated HSF cells was shown to be different from the profile obtained for proteins irradiated at the high-dose (4 Gy). This type of fundamental information regarding radiation-response to cellular events at the molecular level provides a mechanistic basis for identifying relevant molecular markers that can be used in future to better evaluate human health risks at low doses of irradiation and to develop low dose radiation counter measurements.
2006. "Characterization of the Mouse Brain Proteome Using Global Proteomic Analysis Complemented with Cysteinyl-Peptide Enrichment." Journal of Proteome Research 5(2):361-369. Abstract Given the growing interest in applying genomic and proteomic approaches for studying the mammalian brain using mouse models, we hereby present for the first time a comprehensive characterization of the mouse brain proteome. Preparation of the whole brain sample incorporated a highly efficient cysteinyl-peptide enrichment (CPE) technique to complement a global enzymatic digestion method. Both the global and the cysteinyl-enriched peptide samples were analyzed by SCX fractionation coupled with reversed phase LC-MS/MS analysis. A total of 48,328 different peptides were confidently identified (>98% confidence level), covering 7792 non-redundant proteins (~34% of the predicted mouse proteome). 1564 and 1859 proteins were identified exclusively from the cysteinyl-peptide and the global peptide samples, respectively, corresponding to 25% and 31% improvements in proteome coverage compared to analysis of only the global peptide or cysteinyl-peptide samples. The identified proteins provide a broad representation of the mouse proteome with little bias evident due to protein pI, molecular weight, and/or cellular localization. Approximately 26% of the identified proteins with gene ontology (GO) annotations were membrane proteins, with 1447 proteins predicted to have transmembrane domains, and many of the membrane proteins were found to be involved in transport and cell signaling. The MS/MS spectrum count information for the identified proteins was used to provide a measure of relative protein abundances. The mouse brain peptide/protein database generated from this study represents the most comprehensive proteome coverage for the mammalian brain to date, and the basis for future quantitative brain proteomic studies using mouse models.
2006. "Toward Plasma Proteome Profiling with Ion Mobility-Mass Spectrometry." Journal of Proteome Research 5(11):2977-2984. doi:10.1021/pr060232i Abstract Differential, functional, and mapping proteomic analyses of complex biological mixtures suffer from a lack of component resolution. Here we describe the application of ion mobility-mass spectrometry (IMSMS) to this problem. With this approach, components that are separated by liquid chromatography are dispersed based on differences in their mobilities through a buffer gas prior to being analyzed by MS. The inclusion of the gas-phase dispersion provides more than an order of magnitude enhancement in component resolution at no cost to data acquisition time. Additionally, the mobility separation often removes high-abundance species from spectral regions containing low-abundance species, effectively increasing measurement sensitivity and dynamic range. Finally, collision-induced dissociation of all ions can be recorded in a single experimental sequence while conventional MS methods sequentially select precursors. The approach is demonstrated in a single, rapid (3.3 h) analysis of a plasma digest sample where abundant proteins have not been removed. Protein database searches have yielded 731 high confidence peptide assignments corresponding to 438 unique proteins. Results have been compiled into an initial analytical map to be used -after further augmentation and refinement- for comparative plasma profiling studies.
2006. "Mass Measurement Accuracy In Analyses Of Highly Complex Mixtures Based Upon Multidimensional Recalibration." Analytical Chemistry 78(24):8374-8385. doi:10.1021/ac0606251 Abstract Mass spectrometry combined with a range of on-line separation techniques has become a powerful tool for characterization of complex mixtures, including protein digests in proteomics studies. Accurate mass measurements can be compromised due to variations that occur in the course of an on-line separation; e.g. due to excessive space charge in an ion trap, temperature changes, or other sources of instrument “drift”. We have developed a multidimensional recalibration approach that utilizes existing information on the likely mixture composition, taking into account variable conditions of mass measurements, and that corrects the mass calibration for sets of individual peaks binned by e.g. the total ion count for the mass spectrum, the individual peak abundance, m/z value, and liquid chromatography (LC) separation time. The multidimensional recalibration approach uses a statistical matching of measured masses in such measurements, often exceeding 105, to a significant number of putative known species likely to be present in the mixture (i.e. having known accurate masses), to identify a subset of the detected species that serve as effective calibrants. The recalibration procedure involves optimization of the mass accuracy distribution (histogram), to provide a more confident distinction between true and false identifications. We report the mass accuracy improvement obtained for data acquired using a TOF and several FTICR mass spectrometers. We show that the multidimensional recalibration better compensates for systematic mass measurement errors, and also significantly reduces the mass error spread: i.e. both the accuracy and precision of mass measurements are improved. The mass measurement improvement is found to be virtually independent of the initial instrument calibration, allowing e.g. less frequent calibration. We show that this recalibration can provide sub-ppm mass measurement accuracy for measurements of a complex fungal proteome tryptic digest, and provide improved confidence or numbers of peptide identifications.
2006. "Ultra-sensitive and quantitative characterization of proteomes." Molecular Biosystems 2(5):221-230. doi:10.1039/b601468b Abstract Significant technological advances in quantitative proteomics approaches and instrumentation, as well as in related (bio)informatics data analysis have been achieved over the past decade. The so-called “bottom-up” proteomics approach that accounts for nearly all proteomics measurements combines high efficiency separations with MS to characterize highly complex peptide mixtures. Currently the most powerful approach in terms of overall separation power and sensitivity involves gradient elution reversed phase liquid chromatography (LC) coupled to high mass measurement accuracy MS, particularly Fourier transform-ion cyclotron resonance mass spectrometry (FTICR MS). These LC-MS platforms allow large number of peptides to be distinguished. While the dynamic range achievable in any mass spectrum constrains the number of detectable species, the use of separations and their effective integration with MS greatly increases the overall proteomics analysis dynamic range, but at the cost of lower throughput. In this article, we highlight aspects of separations, electrospray ionization (ESI) phenomena, and MS developments that are interrelated and important for making more sensitive and higher throughput quantitative proteomics measurements.
2006. "Future directions for electrospray ionization for biological analysis using mass spectrometry." BioTechniques 41(2):147-148. Abstract Electrospray ionization-mass spectrometry combined with high efficiency capillary liquid chromatography provides the high sensitivity and broad dynamic range measurements important for quantitatively characterizing proteins in complex matrices.
2006. "Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling ." Electrophoresis 27(13):2722-2733. doi:10.1002/elps.200600037 Abstract Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. Two-dimensional gel electrophoresis, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size exclusion chromatography (SEC) fractionation with reversed-phase liquid chromatography (RPLC)-Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry (MS) is compared with the combination of RPLC fractionation with capillary isoelectric focusing (CIEF)-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances.
2006. "High-Resolution Field Asymmetric Waveform Ion Mobility Spectrometry Using New Planar Geometry Analyzers." Analytical Chemistry 78(11):3706-3714. doi:10.1021/ac052020v Abstract Field asymmetric waveform ion mobility spectrometry (FAIMS) has emerged as a powerful tool of broad utility for separation and characterization of gas-phase ions, especially in conjunction with mass spectrometry (MS). In FAIMS, ions are filtered by the dependence of mobility on electric field while carried by gas flow through the analytical gap between two electrodes of either planar (p-) or cylindrical (c-) geometry. Most FAIMS/MS systems employ c-FAIMS because of its ease of coupling to MS, yet the merits of two geometries have not been compared in detail. Here, a priori simulations have revealed that reducing the FAIMS curvature always improves resolution at equal sensitivity. In particular, the resolving power of p-FAIMS exceeds that of c-FAIMS, typically by a factor of 2 - 4 depending on the ion species and carrier gas. We have constructed a new planar FAIMS that incorporates a curtain plate interface for effective operation with an ESI ion source and is joined to MS using an ion funnel interface with a novel slit aperture. The resolution increases up to 4-fold over existing c-FAIMS, even though the analysis is ~3 times faster. This allows separation of species not feasible in previous FAIMS studies, e.g., protonated leucine and isoleucine or new bradykinin isomers. The improvement for protein conformers (of ubiquitin) is less significant, possibly because of multiple unresolved geometries.
2006. "Field Asymmetric Waveform Ion Mobility Spectrometry Studies of Proteins: Dipole Alignment in Ion Mobility Spectrometry?" Journal of Physical Chemistry B 110(43):21966-21980. doi:10.1021/jp062573p Abstract Approaches to characterization and separation of ions involving their mobilities in gases were developed since 1960-s. Conventional ion mobility spectrometry (IMS) measures the absolute mobility and the field asymmetric waveform IMS (FAIMS) exploits the difference between mobilities at high and low electric fields. However, all previous work was based on the orientationally averaged cross-sections Ωavg between ions and buffer gas molecules. Virtually all large ions are electric dipoles that will be oriented by a sufficiently strong electric field. At typical FAIMS conditions, that must happen for dipole moments > ~400 Debye, found for many macroions including most proteins above ~30 kDa. Mobilities of aligned dipoles depend on directional cross-sections Ωdir (rather than Ωavg), which should have a major effect on FAIMS separation parameters. Here we study the FAIMS behavior of ESI-generated ions for ten proteins up to ~70 kDa. Those above 29 kDa exhibit a strong increase of mobility at high field, which is consistent with predicted ion dipole alignment. This effect expands the FAIMS peak capacity by an order of magnitude, allowing separation of up to ~102 distinct protein conformers and revealing information about Ωdir and ion dipole moment that is of potential utility for structural characterization. Possible means to extend the dipole alignment to smaller ions are discussed.
2006. "Feasibility of Higher-Order Differential Ion Mobility Separations Using New Asymmetric Waveforms ." Journal of Physical Chemistry A 110(8):2663-2673. doi:10.1021/jp055349t Abstract Technologies for separating and characterizing ions based on their transport properties in gases have been around for three decades. The early method of ion mobility spectrometry (IMS) distinguished ions by absolute mobility that depends on the collision cross section with buffer gas atoms. The more recent technique of field asymmetric waveform IMS (FAIMS) measures the difference between mobilities at high and low electric fields. Coupling IMS and FAIMS to soft ionization sources and mass spectrometry (MS) has greatly expanded their utility, enabling new applications in biomedical and nanomaterials research. Here, we show that time-dependent electric fields comprising more than two intensity levels could, in principle, effect an infinite number of distinct differential separations based on the higher-order terms of expression for ion mobility. These analyses could employ the hardware and operational procedures similar to those utilized in FAIMS. Methods up to the 4th or 5th order (where conventional IMS is 1st order and FAIMS is 2nd order) should be practical at field intensities accessible in ambient air, with still higher orders potentially achievable in insulating gases. Available experimental data suggest that higher-order separations should be largely orthogonal to each other and to FAIMS, IMS, and MS.
2006. "Characterizing the Structures and Folding of Free Proteins Using 2-D Gas-Phase Separations: Observation of Multiple Unfolded Conformers." Analytical Chemistry 78(10):3304-3315. doi:10.1021/ac060283z Abstract Understanding the 3-D structure and dynamics of proteins and other biological macromolecules in various environments is among the central challenges of chemistry. Electrospray ionization (ESI) can transfer ions from solution to gas phase with only limited structural distortion, allowing their profiling using mass spectrometry and other gas phase approaches. Ion mobility spectrometry (IMS) can be used to separate and characterize macroion conformations with high sensitivity and speed. However, IMS separation power has proven insufficient for full resolution of major structural variants of protein ions and elucidation of their interconversion dynamics. Here we report characterization of macromolecular conformations using field asymmetric waveform IMS (FAIMS) coupled to conventional IMS in conjunction with mass spectrometry. The controlled activation of ions in the electrodynamic funnel trap between FAIMS and IMS stages enables investigating the structural evolution of particular isomeric precursors as a function of the extent and duration of activation that can be varied over a large range. These new capabilities are demonstrated for bovine ubiquitin, a common model for study of structure and folding of gas-phase proteins. For nearly all charge states, two-dimensional FAIMS/IMS separations distinguish many more conformations than either FAIMS or IMS alone, including some species with very low abundance. The unfolding of specific ubiquitin conformers has been studied employing ion heating in the FAIMS/IMS interface.
2006. "Proteomic Analysis of Salmonella enterica Serovar Typhimurium Isolated from RAW 264.7 Macrophages: identification of a novel protein that contributes to the replication of serovar Typhimurium inside macrophages." Journal of Biological Chemistry 281:29131-29140. doi:10.1074/jbc.M604640200 Abstract ABSTRACT: To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time-points following infection and used a liquid chromatography-mass spectrometry (LC-MS)-based proteomic approach to detect the changes in STM protein abundances. Because host resistance to STM infection is strongly modulated by the expression of a functional host resistant regulator, i.e., natural resistance associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely house-keeping proteins whose abundances remain relatively constant during the time-course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of STM cell wall, emphasizing their important roles in the colonization of macrophages by Salmonella.
2006. "Endogenously Nitrated Proteins in Mouse Brain: Links To Neurodegenerative Disease ." Biochemistry 45(26):8009-8022. doi:10.1021/bi060474w Abstract Increased nitrotyrosine modification of proteins has been documented in multiple pathologies in a variety of tissue types; emerging evidence suggests its additional role in redox regulation of normal metabolism. In order to identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic dataset identifying 7,792 proteins from whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in identification of 31 unique nitrotyrosine sites within 29 different proteins. Over half of the nitrated proteins identified have been reported to be involved in Parkinson’s disease, Alzheimer’s disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson’s disease, induces increased nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, characteristics consistent with peroxynitrite-induced tyrosine modification. More striking is the five-fold greater nitration of tyrosines having nearby basic sidechains, suggesting electrostatic attraction of basic groups with the negative charge of peroxynitrite. Together, these results suggest that elevated peroxynitrite generation plays a role in neurodegenerative changes in the brain and provides a predictive tool of functionally important sites of nitration.
2006. "Advances and Challenges in Liquid Chromatography-Mass Spectrometry-Based Proteomics Profiling for Clinical Applications." Molecular & Cellular Proteomics. MCP 5(10):1727-1744. doi:10.1074/mcp.M600162-MCP200 Abstract The advances in proteomic technologies provide tremendous opportunities for applying these technologies in biomarker-related clinical applications; however, the unique characteristics of human biofluids such as high dynamic range in protein abundances and extreme complexity of human proteomes present tremendous challenges for current analytical technologies. In this review, we focus on summarizing the recent advances in LC-MS based proteomic profiling and its applications in clinical proteomics as well as the major challenges for implementing these technologies for more effective biomarker discovery. Over the last few years, tremendous efforts have been directed towards the development of more effective approaches for characterizing the human plasma/serum and other biofluid proteomes. The developments in immunodepletion and various fractionation approaches in combination with much improved LC-MS platforms have enabled the profiling of the plasma proteome with much greater dynamic range of coverage, allowing many proteins at low ng/mL levels being confidently identified. Despite the significant advances and efforts, the dynamic range of measurements or extent of proteome coverage, the confidence of peptide/protein identification, the accuracy of quantitation, the throughput of analysis, and the robustness of the present instrumentation are still among the major challenges for implementation of a proteomic profiling platform suitable for efficient clinical applications.
2006. "Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information." Analytical Chemistry 78(14):5026-5039. doi:10.1021/ac060143p Abstract We describe an improved artificial neural network (ANN)-based method for predicting peptide retention times in reversed phase liquid chromatography. In addition to the peptide amino acid composition, this study investigated several other peptide descriptors to improve the predictive capability, such as peptide length, sequence, hydrophobicity and hydrophobic moment, and nearest neighbor amino acid, as well as peptide predicted structural configurations (i.e., helix, sheet, coil). An ANN architecture that consisted of 1052 input nodes, 24 hidden nodes, and 1 output node was used to fully consider the amino acid residue sequence in each peptide. The network was trained using ~345,000 non-redundant peptides identified from a total of 12,059 LC-MS/MS analyses of more than 20 different organisms, and the predictive capability of the model was tested using 1303 confidently identified peptides that were not included in the training set. The model demonstrated an average elution time precision of ~1.5% and was able to distinguish among isomeric peptides based upon the inclusion of peptide sequence information. The prediction power represents a significant improvement over our earlier report (Petritis et al., Anal. Chem. 2003, 75, 1039-1048) and other previously reported models.
2006. "Quantitative Proteome Analysis of Breast Cancer Cell Lines using 18O-Labeling and an Accurate Mass and Time Tag Strategy." Proteomics 6(9):2903-2915. doi:10.1002/pmic200500582 Abstract Proteome comparison of cell lines derived from breast cancer and normal breast epithelium provide opportunities to identify differentially expressed proteins and pathways associated with specific phenotypes. We employed trypsin-catalyzed 16O/18O peptide labeling, FTI-CR mass spectrometry, and the accurate mass and time (AMT) tag strategy to calculate compare the relative protein abundances of hundreds of proteins simultaneously in non-cancer and cancer cell lines derived from breast tissue. A reference panel of cell lines was created to facilitate comparisons of relative protein abundance amongst multiple cell lines and across multiple experiments. A peptide database generated from multidimensional LC separations and MS/MS analysis was used to facilitate subsequent AMT tag-based peptide identifications. This peptide database represented a total of 2,299 proteins, including 514 that were quantified using the AMT tag and 16O/18O strategies. Eighty-six proteins showed at least a 3-fold protein abundance change between cancer and non-cancer cell lines. A comparison of protein expression profiles with previously published gene expression data revealed that 21 of these proteins also had >3-fold differences between the non-cancer and cancer cell lines at the transcriptional level. Clustering of protein abundance ratios revealed that several groups of proteins were differentially expressed between the cancer cell lines
2006. "Theoretical and experimental evaluation of the low m/z transmission of an electrodynamic ion funnel." Journal of the American Society for Mass Spectrometry 17(4):586-592. Abstract The transmission of ions at low m/z can be either necessary for an application or problematic (e.g. when large numbers of low m/z ions consume a large fraction of an ion trap’s capacity). The low m/z ion transmission limit of an electrodynamic ion funnel has been characterized using both experimental and theoretical approaches. A theoretical model is developed based on a series of infinite wire conductors that represent the ring electrodes of the ion funnel. Mathematical relationships for both low and high m/z cutoffs of the idealized two-dimensional system are derived. The low m/z cutoff is also evaluated through a series of experiments that show it is influenced by both the RF frequency and the DC electric field gradient. However, unlike multipole ion guides, there is no marked dependence of the low m/z cutoff on the RF amplitude, in agreement with theoretical results. With this new understanding, ion funnels can be designed and configured to better match the m/z range requirements for various applications.
2006. "Proteomic approaches to bacterial differentiation ." Journal of Microbiological Methods 67(3):473-486. doi:10.1016/j.mimet.2006.04.024 Abstract While genomic approaches have been applied to the detection and identification of individual bacteria within microbial communities, analogous proteomics approaches have been effectively precluded due to the inherent complexity. An in silico assessment of peptides derived from artificial simple and complex communities was performed to evaluate the effect of proteome complexity on species detection. Detection and validation of predicted peptides initially identified as distinctive within the simple community was experimentally performed using a mass spectrometry-based proteomics approach. An assessment of peptide distinctiveness and the potential for mapping to a particular bacterium within a community was made throughout each step of the study. A second assessment performed in silico of peptide distinctiveness for a complex community of 25 microorganisms was also conducted. The experimental data for a simple community, and the in silico data for a complex community revealed that it is feasible to predict, observe, and quantify distinctive peptides from one organism in the presence of at least a 100-fold greater abundance of another, thus yielding putative markers for the identification of a bacterium of interest. This work represents a first step towards quantitative proteomic characterization of complex microbial communities.
2006. "Proteomic approaches to bacterial differentiation." Journal of Microbiological Methods 67(3):473-486. Abstract While genomic approaches have been applied for the detection and identification of individual bacteria within microbial communities, analogous proteomics approaches have been effectively precluded due to their inherent complexity. An in silico assessment of peptides that could potentially be present in the proteomes of artificial simple and complex communities was performed to evaluate the effect of proteome complexity on species detection. A mass spectrometry-based proteomics approach was employed to experimentally detect and validate the predicted tryptic peptides initially identified as distinctive within the simple community. An assessment of peptide distinctiveness and the potential for mapping to a particular bacterium within a community was made throughout each step of the study. A second in silico assessment of peptide distinctiveness for a complex community of 25 microorganisms was conducted to investigate the levels of instrumental performance that would be required to experimentally detect these peptides, as well as how performance varied with complexity (e.g., the number of different microorganisms). The experimental data for a simple community showed that it is feasible to predict, observe, and to quantify distinctive peptides from one organism in the presence of at least a 100-fold greater abundance of another, thus yielding putative markers for identifying a bacterium of interest. This work represents a first step towards quantitative proteomic characterization of complex microbial communities and the possible development of community wide markers of perturbations to such communities.
2006. "Characterization of the Human Pancreatic Islet Proteome by Two-Dimensional LC/MS/MS." Journal of Proteome Research 5(12):3345-3354. doi: 10.1021/pr060322n Abstract Research to elucidate the pathogenesis of type 1 diabetes mellitus has traditionally focused on the genetic and immunological factors associated with the disease, and, until recently, has not considered the target cell. While there have been reports detailing proteomic analyses of established islet cell lines or isolated rodent islets, the information gained is not always easily extrapolated to humans. Therefore, extensive characterization of the human islet proteome could result in better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the detection of 29,021 unique peptides corresponding to 4,925 proteins. As expected, major islet hormones (insulin, glucagon, somatostatin), beta-cell enriched secretory products (IAPP), ion channels (K-ATP channel), and transcription factors (PDX-1, Nkx 6.1, HNF-1 beta) were detected. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was obtained, including the insulin signaling cascade and the MAP kinase, NF-κβ, and JAK/STAT pathways. This work represents the most extensive characterization of the human islet proteome to date and provides a peptide reference library that may be utilized in future studies of islet biology and type 1 diabetes.
2006. "More sensitive and quantitative proteomic measurements using very low flow rate porous silica monolithic LC columns with electrospray ionization-mass spectrometry ." Journal of Proteome Research 5(5):1091-1097. Abstract The sensitivity of proteomics measurements using liquid chromatography (LC) separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves approximately inversely with liquid flow rate, making attractive the use of smaller inner diameter LC columns. We report the development and initial application of 10 µm i.d. silica-based monolithic LC columns providing more sensitive proteomics measurements. The implementation provides robust performance and suitability for automated proteome analyses due to integration with a micro solid phase extraction pre-column for ease of sample injection and clean-up prior to the reversed phased LC separation. Greater than 10-fold improvement in sensitivity was obtained compared to analyses using more conventional capillary LC, enabling e.g. the identification of >5000 different peptides by MS/MS from 100-ng of a Shewanella oneidensis tryptic digest using an ion trap MS. The low nL/min LC flow rates provide more uniform signal intensities for different peptides, and provided improved quantitative measurements compared to conventional separation systems without the use of internal standards or isotopic labeling. The improved sensitivity allowed LC-MS measurements of immunopurified protein phosphatase 5 that were in good agreement with quantitative western blot analyses.
2006. "High Dynamic Range Characterization of the Trauma Patient Plasma Proteome ." Molecular & Cellular Proteomics. MCP 5(10):1899-1913. doi:10.1074/mcp.M600068-MCP200 Abstract While human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein, we describe a strategy that combines immunoaffinity subtraction and chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with 2D-LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 nonredundant proteins. Numerous low-abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally, a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients, which provides a foundation for future high-throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.
2006. "Evaluation of Multi-Protein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry ." Molecular & Cellular Proteomics. MCP 5(11):2167-2174. Abstract The detection of low-abundance protein disease biomarkers from human blood poses significant challenges due to the high dynamic range of protein concentrations that span more than 10 orders of magnitude, as well as the extreme complexity of the serum/plasma proteome. Therefore, experimental strategies that include the removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum, plasma, and other body fluids to enhance detection of low-abundance proteins and achieve broader proteome coverage. However, both the specificity and reproducibility of the high-abundance protein depletion process represent common concerns. Here, we report a detailed evaluation of the performance of two commercially available immunoaffinity subtraction systems commonly used in human serum/plasma proteome characterization by high resolution LC-MS/MS. One system uses mammalian IgG antibodies to remove six of the most abundant plasma proteins, and the other uses chicken immunoglobulin yolk (IgY) antibodies to remove twelve of the most abundant plasma proteins. Plasma samples were repeatedly processed using these two systems, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. Removal of target proteins by both immunoaffinity subtraction systems proved reproducible and efficient. Nontarget proteins, including spiked protein standards, were also observed to bind to the columns, but in a fairly reproducible manner. The results suggest that these multi-protein immunoaffinity subtraction systems are both highly effective and reproducible for removing high-abundance proteins and therefore, can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies.
2006. "An IMS-IMS Analogue of MS-MS." Analytical Chemistry 78(12):4161-4174. Abstract The development of a new ion mobility/mass spectrometry instrument that incorporates a multifield drift tube/ion funnel design is described. In this instrument, individual components from a mixture of ions can be resolved and selected on the basis of mobility differences prior to collisional activation inside the drift tube. The fragment ions that are produced can be dispersed again in a second ion mobility spectrometry (IMS) region prior to additional collisional activation and MS analysis. The result is an IMS-IMS analogue of MS-MS. Here, we describe the preliminary instrumental design and experimental approach. We illustrate the approach by examining the highly characterized bradykinin and ubiquitin systems. Mobility-resolved fragment ions of bradykinin show that b-type ions are readily discernible fragments, because they exist as two easily resolvable structural types. Current limitations and future directions are briefly discussed.
2006. "PRISM: A Data Management System for High-Throughput Proteomics ." Proteomics 6(6):1783-1790. doi:10.1002/pmic.200500500 Abstract Advanced proteomic research efforts involving areas such as systems biology or biomarker discovery are enabled by the use of high level informatics tools that allow the effective analysis of large quantities of differing types of data originating from various studies. Performing such analyses on a large scale is not feasible without a computational platform that performs data processing and management tasks. Such a platform must be able to provide high-throughput operation while having sufficient flexibility to accommodate evolving data analysis tools and methodologies. The Proteomics Research Information Storage and Management System (PRISM) provides a platform that serves the needs of the accurate mass and time tag approach developed at PNNL. PRISM incorporates a diverse set of analysis tools and allows a wide range of operations to be incorporated by using a state machine that is accessible to independent, distributed computational nodes. The system has scaled well as data volume has increased over several years, while allowing adaptability for incorporating new and improved data analysis tools for more effective proteomics research.
2006. "Chemically Etched Open Tubular and Monolithic Emitters for Nanoelectrospray Ionization Mass Spectrometry." Analytical Chemistry 78(22):7796-7801. Abstract We have developed a new procedure for fabricating fused silica emitters for electrospray ionization-mass spectrometry (ESI-MS) in which the end of a bare fused silica capillary is immersed into aqueous hydrofluoric acid, and water is pumped through the capillary to prevent etching of the interior. Surface tension causes the etchant to climb the capillary exterior, and the etch rate in the resulting meniscus decreases as a function of distance from the bulk solution. Etching continues until the silica touching the hydrofluoric acid reservoir is completely removed, essentially stopping the etch process. The resulting emitters have no internal taper, making them much less prone to clogging compared to e.g. pulled emitters. The high aspect ratios and extremely thin walls at the orifice facilitate very low flow rate operation; stable ESI-MS signals were obtained for model analytes from 5-μm-diameter emitters at a flow rate of 5 nL/min with a high degree of inter-emitter reproducibility. In extensive evaluation, the etched emitters were found to enable approximately four times as many LC-MS analyses of proteomic samples before failing compared with conventional pulled emitters. The fabrication procedure was also employed to taper the ends of polymer monolith-containing silica capillaries for use as ESI emitters. In contrast to previous work, the monolithic material protrudes beyond the fused silica capillaries, improving the monolith-assisted electrospray process.
2006. "Robust Algorithm for Alignment of Liquid Chromatography-Mass Spectrometry Analyses in an Accurate Mass and Time Tag Data Analysis Pipeline." Analytical Chemistry 78(21):7397-7409. Abstract Liquid chromatography coupled to mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) has become a standard technique for analyzing complex peptide mixtures to determine composition and relative quantity. Several high-throughput proteomics techniques attempt to combine complementary results from multiple LC-MS and LC-MS/MS analyses to provide more comprehensive and accurate results. To effectively collate results from these techniques, variations in mass and elution time measurements between related analyses are corrected by using algorithms designed to align the various types of results: LC-MS/MS vs. LC-MS/MS, LC-MS vs. LC-MS/MS, and LC-MS vs. LC-MS. Described herein are new algorithms referred to collectively as Liquid Chromatography based Mass Spectrometric Warping and Alignment of Retention times of Peptides (LCMSWARP) which use a dynamic elution time warping approach similar to traditional algorithms that correct variation in elution time using piecewise linear functions. LCMSWARP is compared to a linear alignment algorithm that assumes a linear transformation of elution time between analyses. LCMSWARP also corrects for drift in mass measurement accuracies that are often seen in an LC-MS analysis due to factors such as analyzer drift. We also describe the alignment of LC-MS results and provide examples of alignment of analyses from different chromatographic systems to demonstrate more complex transformation functions.
2006. "Improving Mass Spectrometer Sensitivity Using a High-Pressure Electrodynamic Ion Funnel Interface." Journal of the American Society for Mass Spectrometry 17(9):1299-1305. doi:10.1016/j.jasms.2006.06.005 Abstract We report on a new electrodynamic ion funnel that operates at a pressure of 30 Torr with no loss of ion transmission. The enhanced performance compared to previous ion funnel designs optimized for pressures of <5 Torr was achieved by reducing the ion funnel capacitance and increasing the RF drive frequency (1.7 MHz) and amplitude (100-170 V peak-to-peak). No degradation of ion transmission was observed for pressures from 2 - 30 Torr. The ability to operate at higher pressure enabled a new dual ion funnel mass spectrometer (MS) interface design that can accommodate a greater gas load. When combined with a multicapillary inlet, the interface provided more efficient introduction of ions, resulting in a significant enhancement in MS sensitivity and detection limits.
2006. "Biomarker Candidate Identification in Yersinia Pestis Using Organism-Wide Semiquantitative Proteomics ." Journal of Proteome Research 5(11):3008-3017. Abstract Yersinia pestis, the causative agent of plague, is listed by the CDC as a level A select pathogen. To better enable detection, intervention and treatment of Y. pestis infections, it is necessary to understand its protein expression under conditions that promote or inhibit virulence. To this end, we have utilized a novel combination of the accurate mass and time tag methodology of mass spectrometry and clustering analysis using OmniViz™ to compare the protein abundance changes of 992 identified proteins under four growth conditions. Temperature and Ca2+ concentration were used to trigger virulence associated protein expression fundamental to the low calcium response. High-resolution liquid chromatography and electrospray ionization mass spectrometry were utilized to determine protein identity and abundance on the genome-wide level. The cluster analyses revealed, in a rapid visual platform, the reproducibility of the current method as well as relevant protein abundance changes of expected and novel proteins relating to a specific growth condition and sub-cellular location. Using this method, 89 proteins were identified as having a similar abundance change profile to 29 known virulence associated proteins, providing additional biomarker candidates for future detection and vaccine development strategies.
2006. "Differential Label-free Quantitative Proteomic Analysis of Shewanella oneidensis Cultured under Aerobic and Suboxic Conditions by Accurate Mass and Time Tag Approach." Molecular & Cellular Proteomics. MCP 5(4):714-725. doi:10.1074/mcp.M500301-MCP200 Abstract We describe the application of liquid chromatography coupled to mass spectrometry (LC/MS) without the use of stable isotope labeling for differential quantitative proteomics analysis of whole cell lysates of Shewanella oneidensis MR-1 cultured under aerobic and sub-oxic conditions. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to initially identify peptide sequences, and LC coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) was used to confirm these identifications, as well as measure relative peptide abundances. 2343 peptides, covering 668 proteins were identified with high confidence and quantified. Among these proteins, a subset of 56 changed significantly using statistical approaches such as SAM, while another subset of 56 that were annotated as performing housekeeping functions remained essentially unchanged in relative abundance. Numerous proteins involved in anaerobic energy metabolism exhibited up to a 10-fold increase in relative abundance when S. oneidensis is transitioned from aerobic to sub-oxic conditions.
2006. "“Zero-length” Cross-linking in Solid State as an Approach for Analysis of Protein -Protein Interactions." Protein Science 15(3):429-440. Abstract Analyzing the architecture of protein complexes is a difficult task. Chemical cross-linking is often used in combination with mass spectrometric analysis to elucidate the interaction interfaces between proteins. We have developed a new approach for the analysis of interacting interfaces in protein complexes based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum and cross-links are introduced in the solid phase by dehydrating the protein in a non-water solvent, thus, creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H216O and H218O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C-terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides.We demonstrated our method by cross-linking homooligomeric protein complexes alone or in a mixture of many other proteins. Cross-linking in the solid state was shown to be specific and reproducible. Glutathione-S-transferase (GST) from Schistosoma japonicum was studied in more detail. Twenty-seven unique intra-molecular and two inter-molecular cross-linked peptides were identified using tryptic mapping followed by LTQ-MS analysis. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. Identified cross-linked peptides were validated by computational (visualization of cross-links in the three-dimensional [3D] structure of GST) and experimental (MS/MS) analyses. Most of the identified cross-links matched interacting peptides in the native 3D structure of GST indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru(II)Bpy32+ catalyzed, oxidative “zero-length” cross-linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.
2006. "Confirmation of the Expression of a Large Set of Conserved Hypothetical Proteins in Shewanella oneidensis MR-1." Journal of Microbiological Methods 66(2):223-233. Abstract High-throughput “omic” technologies have allowed for a relatively rapid, yet comprehensive analysis of the global expression patterns within an organism in response to perturbations. In the current study, tryptic peptides were identified with high confidence from capillary liquid chromatography-mass spectrometry analysis of 26 chemostat cultures of Shewanella oneidensis MR-1 under various conditions. Using at least one distinctive and a total of two total peptide identifications per protein, we detected the expression of 758 conserved hypothetical proteins. This included 359 such proteins previously described (Kolker et al, 2005) with an additional 399 reported herein for the first time. The latter 399 proteins ranged from 5.3 to 208.3 kDa, with 44 being of 100 amino acid residues or less. Using a combination of information including peptide detection in cells grown under specific culture conditions and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some conserved hypothetical proteins. Such proteins were found not only to be expressed, but 19 were only expressed under certain culturing conditions, thereby providing insight into potential functions. These findings also impact the genomic annotation for S. oneidensis MR-1 by confirming that these genes code for expressed proteins. Our results indicate that 399 proteins can now be upgraded from “conserved hypothetical protein” to “expressed protein in Shewanella,” 19 of which appeared to be expressed under specific culture conditions.
2006. "The Proteome of Dissimilatory Metal-reducing Microorganism Geobacter Sulfurreducens under Various Growth Conditions." Biochimica et Biophysica Acta--Proteins and Proteomics 1764(7):1198-1206. doi:10.1016/j.bbapap.2006.04.017 Abstract The global protein analysis of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under eight different growth conditions in order to enhance the potential that genes would be expressed. Over 3,187 gene products, representing about 92% of the total predicted gene products in the genome, were detected. The AMT approach was able to identify a much higher number of proteins than could be detected with the 2-D PAGE approach. A high proportion of predicted proteins in most protein role categories were detected with the highest number of proteins identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Localization studies indicated that computational predictions of cytochrome location were limited. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.
2006. "HepatoProteomics: Applying Proteomic Technologies to the Study of Liver Function and Disease." Hepatology 44(2):299-308. Abstract The wealth of human genome sequence information now available, coupled with technological advances in robotics, nanotechnology, mass spectrometry, and information systems, has given rise to a method of scientific inquiry known as functional genomics. By using these technologies to survey gene expression and protein production on a near global scale, the goal of functional genomics is to assign biological function to genes with currently unknown roles in physiology. This approach carries particular appeal in disease research, where it can uncover the function of previously unknown genes and molecular pathways that are directly involved in disease progression. With this knowledge may come improved diagnostic techniques, prognostic capabilities, and novel therapeutic approaches. In this regard, the continuing evolution of proteomic technologies has resulted in an increasingly greater impact of proteome studies in many areas of research and hepatology is no exception. Our laboratory has been extremely active in this area, applying both genomic and proteomic technologies to the analysis of virus-host interactions in several systems, including the study of hepatitis C virus (HCV) infection and HCV-associated liver disease. Since proteomic technologies are foreign to many hepatologists (and to almost everyone else), this article will provide an overview of proteomic methods and technologies and describe how they’re being used to study liver function and disease. We use our studies of HCV infection and HCV-associated liver disease to present an operational framework for performing high throughput proteome analysis and extracting biologically meaningful information.
2006. "Normalization Approaches for Removing Systematic Biases Associated with Mass Spectrometry and Label-Free Proteomics." Journal of Proteome Research 5(2):277-286. doi:10.1021/pr050300l Abstract Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias, assigned ranks among the techniques revealed significant trends. For most LC-FTICR MS analyses, linear regression normalization ranked either first or second among the four techniques, suggesting that this technique was more generally suitable for reducing systematic biases.
2006. "Comparison of aerobic and photosynthetic Rhodobacter sphaeroides 2.4.1 proteomes." Journal of Microbiological Methods 67(3):424-436. doi:10.1016/j.mimet.2006.04.021 Abstract Proteomes from aerobic and photosynthetic grown Rhodobacter sphaeroides 2.4.1 cell cultures were characterized using liquid chromatography-mass spectrometry in conjunction with an accurate mass and elution time (AMT) tag approach. Roughly 8000 high quality peptides were detected that represented 1,445 gene products and 34% of the predicted proteins. The identified proteins corresponded primarily to open reading frames (ORFs) contained within the two chromosomal elements of this bacterium, but a significant number were also observed from ORFs associated with 5 naturally occurring plasmids. Data mining of peptides revealed a number of proteins uniquely detected within the photosynthetic cell culture. Proteins observed in both aerobic respiratory and photosynthetic grown cultures were analyzed semi-quantitatively by comparing their estimated abundances to provide insights into bioenergetic models for aerobic respiration and photosynthesis. Additional emphasis was placed on gene products annotated as hypothetical to gain information as to their potential roles within these two growth conditions. Where possible, transcriptome data for R. sphaeroides obtained under the same culture conditions were compared with these results. This comparative study demonstrated the applicability of the AMT tag approach for high-throughput proteomic analyses of pathways associated with the photosynthetic lifestyle.
2006. "Application of the Accurate Mass and Time Tag Approach to the Proteome Analysis of Sub-cellular Fractions Obtained from Rhodobacter sphaeroides 2.4.1 Aerobic and Photosynthetic Cell Cultures." Journal of Proteome Research 5(8):1940-1947. Abstract Abstract The high-throughput accurate mass and time tag (AMT) proteomic approach was utilized to characterize the proteomes for cytoplasm, cytoplasmic membrane, periplasm, and outer membrane fractions from aerobic and photosynthetic cultures of the gram-nagtive bacterium Rhodobacter sphaeroides 2.4.1. In addition, we analyzed the proteins within purified chromatophore fractions that house the photosynthetic apparatus from photosynthetically grown cells. In total, 8300 peptides were identified with high confidence from at least one sub-cellular fraction from either cell culture. These peptides were derived from 1514 genes or 35% percent of proteins predicted to be encoded by the genome. A significant number of these proteins were detected within a single sub-cellular fraction and their localization was compared to in-silico predictions. However, the majority of proteins were observed in multiple sub-cellular fractions, and the most likely sub-cellular localization for these proteins was investigated using a Z-score analysis of peptide abundance along with clustering techniques. Good (81%) agreement was observed between the experimental results and in-silico predictions. The AMT tag approach provides localization evidence for those proteins that have no predicted localization information, those annotated as putative proteins, and/or for those proteins annotated as hypothetical and conserved hypothetical.
2006. "Integrated Molecular Signature of Disease: Analysis of Influenza Virus-Infected Macaques through Functional Genomics and Proteomics." Journal of Virology 80(21):10813 (16 pages). doi:10.1128/JVI.00851-06 Abstract Recent outbreaks of avian influenza in humans have stressed the need for an improved non-human primate model of influenza pathogenesis. In order to develop our macaque model, we expanded our in vivo and functional genomics experiments: We focused on the innate immune response at day 2 post-inoculation and on gene expression in affected lung tissue with viral genetic material present; finally, we sought to identify signature genes for early infection in whole blood. For these purposes, we infected six pigtailed macaques with 107 TCID50 of influenza A/Texas/36/91 virus and three control animals with a sham inoculate. We sacrificed one control and two experimental animals at day 2, 4, and 7 and lung tissue was harvested for pathology, gene expression profiling, and proteomics. Additionally, blood was collected for genomics every other day from each animal until its endpoint. Gross and microscopic pathology, immunohistochemistry, viral gene expression by arrays and/or quantitative real-time RT-PCR confirmed successful yet mild infection in all experimental animals. Genomic experiments were performed using second generation macaque-specific oligonucleotide arrays and high-throughput proteomics revealed host response to infection at the protein level. Our data showed dramatic differences in gene expression within the same influenza-induced lesion based on the presence or absence of viral mRNA. We also identified genes tightly co-regulated in peripheral white blood cells and in lung tissue at day 2 post-inoculation. This latter finding opens the possibility of using gene expression arrays on whole blood to detect infection after exposure but prior to onset of symptoms or shedding.
2006. "Analysis of the Salmonella typhimurium Proteome through Environmental Response toward Infectious Conditions." Molecular & Cellular Proteomics. MCP 5(8):1450-1461. doi:10.1074/mcp.M600139-MCP200 Abstract Salmonella enterica serovar Typhimurium (aka, S. typhimurium) is a facultative intracellular pathogen that causes ~40,000 reported cases of acute gastroenteritis and diarrhea a year in the United States. To develop a deeper understanding of the infectious state of S. typhimurium, liquid chromatography-mass spectrometry-based “bottom-up” proteomics was used to globally analyze the proteins present under specific growth conditions. Salmonella typhimurium LT2 strain cells were grown in contrasting culture conditions that mimicked both natural free-living conditions and an infectious state, i.e., logarithm phase, stationary phase and Mg-depleted medium growth. Initial comparisons of the LT2 strain protein abundances among cell culture conditions indicate that the majority of proteins do not change significantly. Not unexpectedly, cells grown in Mg-depleted medium conditions had a higher abundance of Mg2+ transport proteins than found in other growth conditions. A second more virulent Salmonella typhimurium strain (14028) was also studied with these growth conditions and used to directly compare to the LT2 strain. The strain comparison offers a unique opportunity to compare and contrast observations in these closely related bacteria. One particular protein family, propanediol utilization proteins, was drastically more abundant in the 14028 strain than in the LT2 strain, and may be a contributor to increased pathogenicity in the 14028 strain.
2005. "High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry ." Molecular & Cellular Proteomics. MCP 4:144-155. Abstract It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.
2005. "High Throughput Quantitative Analysis of Serum Proteins using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry." Molecular & Cellular Proteomics. MCP 4(2):144-155. Abstract It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease, and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible and robust to detect potential biomarkers below the level of highly expressed proteins, to generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. In this paper, we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these, no de-glycosylated peptides by LC-ESI-MS, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared to their control littermates.
2005. "Characterization of purified c-type heme-containing peptides and identification of c-type heme-attachment sites in Shewanella oneidenis cytochromes using mass spectrometry." Journal of Proteome Research 4(3):846-854. doi:10.1021/pr0497475 Abstract We describe methods for mass spectrometric identification of heme-containing peptides from digests of c-type cytochromes that contain the CXXCH(X = any amino acid) sequence motif. Analysis of purified standard heme-containing peptides showed that the charged heme group was present both before and after peptide fragmentation in the gas phase. The heme fragment ion yielded the most abundant MS/MS peak for standard heme-containing peptides with one amino acid difference (DAA=1) for both 2+ and 3+ peptide charge states and the extent of heme loss during peptide fragmentation was affected by both sequence and charge. A modified search strategy was evaluated with tryptic digests of one known and two unknown cytochromes from Shewanella oneidenis, demonstrating that this approach can be generally applied for identification of c-type heme-containing peptides from complex samples.
2005. "Development and Evaluation of a Micro- and Nanoscale Proteomic Sample Preparation Method." Journal of Proteome Research 4(6):2397-2403. doi:10.1021/pr050160f Abstract Efficient and effective sample preparation of micro- and nano-scale (micro- and nano-gram) clinical specimens for proteomic applications is often difficult due to losses during the processing steps. Herein we describe a simple “single-tube” preparation protocol appropriate for small proteomic samples using the organic co-solvent, trifluoroethanol (TFE). TFE facilitates both protein extraction and protein denaturation without requiring a separate cleanup step, thus minimizing sample loss. The performance of the TFE method was initially evaluated by comparing to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE protocol provided comparable results to the traditional detergent-based protocols for larger samples (milligrams), based on both sample recovery and peptide/protein identification. The effectiveness of this protocol for micro- and nano-scale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (~ 20 g total protein content) and also for samples of ~ 5 000 human breast cancer MCF-7 cells (~ 500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.
2005. "Two-Dimensional Gas-Phase Separations Coupled to Mass Spectrometry for Analysis of Complex Mixtures." Analytical Chemistry 77(19):6381-6388. Abstract Ion mobility spectrometry (IMS) has been explored for decades, and its versatility in separation and identification of gas-phase ions, including in isomeric mixtures, is well established. Recently, field asymmetric waveform IMS (FAIMS) has been gaining acceptance in similar applications. Coupled to mass spectrometry (MS), both IMS and FAIMS have shown the potential for broad utility in proteomics and other biological analyses. A major attraction of these separations is extremely high speed, exceeding that of condensed-phase alternatives by orders of magnitude. However, modest separation peak capacities have limited the utility of FAIMS and IMS for analyses of complex mixtures. We report 2-D gas-phase separations that join FAIMS to IMS, in conjunction with high-resolution and accuracy time-of-flight MS. Evaluation of FAIMS/IMS/TOF performance using a protein mixture tryptic digest reveals high orthogonality between FAIMS and IMS dimensions, and hence the benefit of FAIMS filtering prior to IMS/MS. The effective overall peak capacities are ~500 for FAIMS/IMS separations, and ~106 for 3-D FAIMS/IMS/MS analyses of tryptic peptides. Implementation of FAIMS/IMS and IMS/MS interfaces using electrodynamic ion funnels greatly improves sensitivity, making FAIMS/IMS/MS a potential platform for ultrahigh-throughput analyses of complex mixtures.
2005. "High-Sensitivity Ion Mobility Spectrometry/Mass Spectrometry Using Electrodynamic Ion Funnel Interfaces ." Analytical Chemistry 77(10):3330-3339. doi:10.1021/ac048315a Abstract The utility of ion mobility spectrometry (IMS) for separation of mixtures and structural characterization of ions has been demonstrated extensively, including in the biological and nanoscience contexts. A major attraction of IMS is its speed, several orders of magnitude above that of condensed-phase separations. Nonetheless, IMS combined with mass spectrometry (MS) has remained a niche technique, substantially due to limited sensitivity resulting from ion losses at the IMS-MS junction. We have developed a new electrospray ionization (ESI)-IMS-QToF MS instrument that incorporates electrodynamic ion funnels at both front ESI-IMS and back IMS-QToF interfaces. The front funnel is of the novel “hourglass” design that efficiently accumulates ions and pulses them into the IMS drift tubes. Even for drift tubes of two meter length, ion transmission through IMS and on to QToF is essentially lossless across the range of ion masses relevant to most applications. The RF ion focusing at IMS terminus does not degrade IMS resolving power, which exceeds 100 (for singly-charged ions) and is close to the theoretical limit. The overall sensitivity of present ESI-IMS-MS system is shown to be comparable to that of commercial ESI-MS, which should make IMS-MS suitable for analyses of complex mixtures with ultra-high sensitivity and exceptional throughput.
2005. "Combining Capillary Electrophoresis with Mass Spectrometry for Applications in Proteomics." Electrophoresis 26(7-8):1291-1305. Abstract Throughout the field of global proteomics, ranging from simple organism studies to human medical applications, the high sample complexity creates demands for improved separations and analysis techniques. Furthermore, with increased organism complexity, the correlation between proteome and genome becomes less certain due to extensive mRNA processing prior to translation. In this way, the same DNA sequence can potentially code for regions in a number of distinct proteins; quantitative differences in expression (or abundance) between these often-related species are of significant interest. Well-established proteomics techniques, which use genomic information to identify peptides that originate from protease digestion, often cannot easily distinguish between such gene products; intact protein-level analyses are required to complete the picture, particularly for identifying post-translational modifications. While chromatographic techniques are currently better suited to peptide analysis, capillary electrophoresis (CE) in combination with mass spectrometry (MS) may become important for intact protein analysis. This review focuses on CE/MS instrumentation and techniques showing promise for such applications, highlighting those with greatest potential. Reference will also be made to developments relevant to peptide-level analyses for use in time- or sample-limited situations.
2005. "Optimization of the Design and Operation of FAIMS Analyzers." Journal of the American Society for Mass Spectrometry 16(1):2-12. Abstract Field Asymmetric waverform Ion Mobility Spectrometry (FAIMS) holds significant promise for post-ionization separations in conjunction with mass-spectrometric analyses. Since commercial systems became available, applications of FAIMS/MS have expanded in scope. However, a relatively poor understanding of fundamentals of FAIMS analyzers has made their design and operation an essentially empirical exercise. Recently we developed a first-principles simulation of FAIMS that accounts for both ion diffusion (including high-field and anisotropic components) and Coulomb repulsion. This model has been validated by extensive comparisons with FAIMS/MS data. Here we further corroborate it by FAIMS-only measurements, and apply it to explore how key instrumental parameters (analytical gap width and length, waveform frequency and profile, the buffer gas identity, and gas flow speed) affect FAIMS response. We find that the trade-off between resolution and sensitivity can be managed by varying gap width, RF frequency, and (in certain cases) buffer gas, with equivalent outcome. The maximum resolving power attainable is ~ 30 - 40. Throughput may be increased by either accelerating the gas flow (preferable) or shortening the device, but at some point the performance starts deteriorating. Resolution and/or sensitivity would be improved by switching from sinusoidalbased to retangular waveforms. For both, the ratio of two between voltages at "high" and "low" parts of the cycle produces best performance.
2005. "FAIMS Operation for Realistic Gas Flow Profile and Asymmetric Waveforms Including Electronic Noise and Ripple." Journal of the American Society for Mass Spectrometry 16(9):1447-1455. doi:10.1016/j.jasms.2005.04.003 Abstract The use of Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) has rapidly grown with the advent of commercial FAIMS systems coupled to mass spectrometry. However, many fundamental aspects of FAIMS remain obscure, hindering its technological improvement and expansion of analytical utility. Recently, we developed a comprehensive numerical simulation approach to FAIMS that can handle any device geometry and operational conditions. The formalism was originally set up in one dimension for a uniform gas flow and limited to ideal asymmetric voltage waveforms. Here we extend the model to account for a realistic gas flow velocity distribution in the analytical gap, axial ion diffusion, and waveform imperfections (e.g. noise and ripple). The non-uniformity of gas flow velocity profile has only a minor effect, slightly improving resolution. However, waveform perturbations are significant even at very low levels, in some cases ~ 0.01% of nominal voltage. These perturbations always improve resolution and decrease sensitivity. Variation of noise or ripple amplitude produces a trade-off between resolution and sensitivity. This trade-off is physically equivalent to that obtained via adjustment of the gap width and/or asymmetric waveform frequency, but the scaling of low-frequency ripple appears to be a more practical way to control FAIMS resolution.
2005. "Making broad proteome protein measurements in 1-5 min using high-speed RPLC separations and high-accuracy mass measurments." Analytical Chemistry 77(23):7763-7773. doi: 10.1021/ac051257o Abstract The throughput for proteomics measurements that provide broad protein coverage is limited by the quality and speed of both the separations and the subsequent mass analysis; present analysis times can range anywhere from hours to days (or longer). We have explored the basis for ultrahigh-throughput proteomics measurements using high-speed reversed-phase liquid chromatography (RPLC) combined with high accuracy mass spectrometric measurements. Time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometers were evaluated in conjunction with 0.8-µm porous C18 particle-packed RPLC using 50 µm i.d. capillary columns for identifying peptides using the Accurate Mass and Time (AMT) tag approach. Peptide RPLC relative retention (elution) times could be correlated to within 5% to elution times that differed by at least 25-fold in speed, which allowed peptides to be identified using AMT tags identified from much slower RPLC-MS/MS analyses. When coupled with RPLC, the mass spectrometers operated at fast spectrum acquisition speeds (e.g., 0.2 sec for TOF and either 0.3 or 0.6 sec for FTICR), and peptide mass measurement accuracies of better than ±15 ppm were obtained. Ion population control during fast separations limited the mass accuracies obtained with FTICR, but the use of fast regulation of ion populations using automated gain control improved the mass accuracies. The detection of low abundance species was somewhat suppressed for fast analyses. The proteome coverage obtained using AMT tags was limited by the separation peak capacity, the sensitivity of the MS, and the accuracy of both the mass measurements and the relative RPLC peptide elution times. Experimental results demonstrated that accuracies of 5% for the RPLC relative elution times and better than ±15 ppm for mass measurements were sufficient for confident identification of >2800 peptides and >760 proteins from >13,000 different detected species from a Shewanella oneidensis tryptic digest.. The TOF instrumentation was found to be preferable for faster separations (of <120 sec), while FTICR MS was more effective for analysis times of >150 sec due to the improved mass accuracies achievable with longer spectrum acquisition times. The present work demonstrates the feasibility of very high throughput proteomics measurements and indicates additional significant improvements in throughput are achievable by further increasing the speed of high peak capacity separations, as well by increasing the measurement sensitivity and the accuracy of mass measurements.
2005. "Characterization of the human blood plasma proteome." Proteomics 5(15):4034-4045. Abstract We describe methods for broad characterization of the human plasma proteome. The combination of stepwise IgG and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of >94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (<30 pg/mL to ~30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin. The results from this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies for the identification of novel protein disease markers, as well as further studies of protein-protein interactions.
2005. "Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics ." Analytical Chemistry 77(10):3090-3100. Abstract Proteomics analysis based-on liquid chromatography (LC), particularly reversed-phase LC (RPLC), is widely practiced; however, cutting-edge LC performance variations have generally not been adopted even though their benefits are well established. The two major reasons behind this general underutilization are: 1) uncertainties surrounding the extent of improvement (e.g., proteome coverage), and 2) the lack of availability of automated, robust, and convenient LC instrumentation. Here, we describe an automated format 20K psi gradient nanoscale LC system that was developed to provide improved separations and sensitivity for proteomics (and metabolomics) applications. The system includes on-line coupling of micro solid phase extraction for sample loading and allows emitters for electrospray ionization to be readily replaced. The system uses 40 to 200 cm 50 µm i.d. fused silica capillaries packed with 1.4- to 3-µm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1,000-1,500 for complex peptide and metabolite mixtures. This separation quality allowed high confidence identification of >12,000 different peptides from >2,000 distinct Shewanella oneidensis proteins (~ 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The reproducibility was >87% for proteins identified between replicates. The protein MS/MS identification rate average exceeded 10 proteins per minute, e.g., 1,207 proteins were identified in 120 min through assignment of 5,944 different peptides. For a human blood plasma sample that was not depleted of the most abundant proteins, 835 distinct proteins were identified with high confidence in a single 12-h run. A single run with accurate mass MS detected >5,000 different compounds from a metabolomics sample.
2005. "Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics." Expert Review of Proteomics 2(3):431-447. Abstract We review recent development in separations and mass spectrometric instrumentation for sensitive and high-throughput proteomic analyses. These efforts have been primarily focused on the development of high-efficiency (separation peak capacity of ~103) nanoscale liquid chromatography (nanoLC; e.g., flow rates extending down to ~20 nL/min at optimal separation linear velocities through narrow packed capillaries) in combination with advanced mass spectrometry (MS), including high sensitivity and high resolution Fourier transform ion cyclotron resonance (FTICR) MS. This technology enables MS analysis of low nanogram-level proteomic samples (i.e., nanoscale proteomics) with individual protein identification sensitivity at the low zeptomole-level. The resultant protein measurement dynamic range can reach 106 for nanogram-sized proteomic samples, while more abundant proteins can be detected from complex sub-picogram size proteome samples. The average proteome identification throughput using MS/MS is >200 proteins/h for a ~3 h analysis. These qualities provide the foundation for proteomics studies of single or small populations of cells. The instrumental robustness required for automation and providing high quality routine performance nanoscale proteomic analyses is also discussed.
2005. "Global whole-cell FTICR mass spectrometric proteomics analysis of the heat shock response in the radioresistant bacterium Deinococcus radiodurans." Journal of Proteome Research 4(3):709-718. Abstract Despite intense interest in the response to radiation in D. radiodurans, little is known about how the organism responds to other stress factors. Our previous studies indicated that D. radiodurans mounts a regulated protective response to heat shock, and that expression of the groESL and dnaKJ operons are induced in response to elevated temperature. In order to gain greater insight into the heat shock response of D. radiodurans on a more global scale, we undertook the study reported here. Using whole-cell semiquantitative mass spectrometric proteomics integrated with global transcriptome microarray analyses, we have determined a core set of highly induced heat shock genes whose expression correlates well at the transcriptional and translational levels. In addition, we observed that the higher the absolute expression of a given gene at physiological conditions, the better the quantitative correlation between RNA and protein expression levels.
2005. "Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples." Electrophoresis 26(14):2729-2738. Abstract Laser microdissection (LM) allows the collection of homogeneous tissue- and cellspecific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.
2005. "Quantitative Proteome Analysis of Human Plasma Following in vivo Lipopolysaccharide Administration using O-16/O-18 Labeling and the Accurate Mass and Time Tag Approach." Molecular & Cellular Proteomics. MCP 4(5):700-709. Abstract Identification of novel diagnostic or therapeutic biomarkers from human blood plasma would benefit significantly from quantitative measurements of the proteome constituents over a range of physiological conditions. We describe here an initial demonstration of proteome-wide quantitative analysis of human plasma. The approach utilizes post-digestion trypsin-catalyzed 16O/18O labeling, two-dimensional liquid chromatography (LC)-Fourier transform ion cyclotron resonance ((FTICR) mass spectrometry, and the accurate mass and time (AMT) tag strategy for identification and quantification of peptides/proteins from complex samples. A peptide mass and time tag database was initially generated using tandem mass spectrometry (MS/MS) following extensive multidimensional LC separations and the database serves as a ‘look-up’ table for peptide identification. The mass and time tag database contains >8,000 putative identified peptides, which yielded 938 confident plasma protein identifications. The quantitative approach was applied to the comparative analyses of plasma samples from an individual prior to and 9 hours after lipopolysaccharide (LPS) administration without depletion of high abundant proteins. Accurate quantification of changes in protein abundance was demonstrated with both 1:1 labeling of control plasma and the comparison between the plasma samples following LPS administration. A total of 429 distinct plasma proteins were quantified from the comparative analyses and the protein abundances for 28 proteins were observed to be significantly changed following LPS administration, including several known inflammatory response mediators.
2005. "Probability-Based Evaluation of Peptide and Protein Identifications from Tandem Mass Spectrometry and SEQUEST Analysis: The Human Proteome." Journal of Proteome Research 4(1):53-62. Abstract Large scale protein identifications from highly complex protein mixtures have recently been achieved using multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) and subsequent database searching with algorithms such as SEQUEST. Here, we describe a probability-based evaluation of false positive rates associated with peptide identifications from three different human proteome samples. Peptides from human plasma, human mammary epithelial cell (HMEC) lysate, and human hepatocyte (Huh)-7.5 cell lysate were separated by strong cation exchange (SCX) chromatography coupled offline with reversed-phase capillary LC-MS/MS analyses. The MS/MS spectra were first analyzed by SEQUEST, searching independently against both normal and sequence-reversed human protein databases, and the false positive rates of peptide identifications for the three proteome samples were then analyzed and compared. The observed false positive rates of peptide identifications for human plasma were significantly higher than those for the human cell lines when identical filtering criteria were used, which suggests that the false positive rates are highly dependent on sample characteristics, particularly the number of proteins found within the detectable dynamic range. Two new sets of filtering criteria are proposed for human plasma and human cell lines, respectively, to provide an overall confidence of >95% for peptide identifications. The new criteria were compared, using a normalized elution time (NET) criterion (Petritis et al. Anal. Chem. 2003, 75, 1039-48), with previously published criteria (Washburn et al. Nat. Biotechnol. 2001, 19, 242-7). The results demonstrate that the present criteria provide significantly higher levels of confidence for peptide identifications.
2005. "Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry." Proteomics 5(2):572-584. doi:10.1002/pmic.200400942 Abstract There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 1563 distinct plasma proteins were confidently identified with 26 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators, and thus constitute potential biomarkers for inflammatory response.
2005. "Comparative Proteome Analyses of Human Plasma Following in vivo Lipopolysaccharide Administration Using Multidimensional Separations Coupled with Tandem Mass Spectrometry." Proteomics 5(2):572-584. Abstract There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator and von Willebrand factor, and thus constituting potential biomarkers for inflammatory response.
2005. "Comparison of Normal and Breast Cancer Cell lines using Proteome, Genome and Interactome data." Journal of Proteome Research 4(6):1952-1960. Abstract Normal and cancer cell line proteomes were profiled using high throughput mass spectrometry techniques. Application of both protein-level and peptide-level sample fractionation combined with LC-MS/MS analysis enabled the confident identification of 2,235 unmodified proteins representing a broad range of functional and compartmental classes. An iterative multi-step search strategy was used to identify post-translational modifications and detected several proteins that are preferentially modified in cancer cells. Information regarding both unmodified and modified protein forms was combined with publicly available gene expression and protein-protein interaction data. The resulting integrated dataset revealed several functionally related proteins that are differentially regulated between normal and cancer cell lines.
2005. "Variable low-mass filtering using an electrodynamic ion funnel." Journal of Mass Spectrometry 40(9):1215-1222. doi:10.1002/jms.900 Abstract An adjustable, low mass-to-charge (m/z) filter has been developed and demonstrated using electrospray ionization to block ions associated with unwanted low m/z species from entering the mass spectrometer and contributing their space charge to down-stream ion accumulation steps. The low-mass filter is made by using an adjustable potential energy barrier from the conductance limiting terminal electrode of an electrodynamic ion funnel, which prohibits species with higher ion mobilities from being transmitted. We show that this arrangement provides a linear voltage adjustment of low-mass filtering from m/z 50 to 500. Mass filtering above m/z 500 can also be performed; however, higher m/z species are attenuated. The mass filter was evaluated with a liquid chromatography-mass spectrometry analysis of an albumin tryptic digest and resulted in the ability to block low-mass, “background” ions which account for 40-70% of the total ion current from the ESI source during peak elution.
2005. "Automatic Gain Control in Mass Spectrometry using a Jet Disrupter Electrode in an Electrodynamic Ion Funnel." Journal of the American Society for Mass Spectrometry 16(2):244-253. Abstract We report on the use of a jet disrupter electrode in an electrodynamic ion funnel as an electronic valve to regulate the intensity of the ion beam transmitted through the interface of a mass spectrometer in order to perform automatic gain control (AGC). The ion flux is determined by either directly detecting the ion current on the conductance limiting orifice of the ion funnel or using a short mass spectrometry acquisition. Based upon the ion flux intensity, the voltage of the jet disrupter is adjusted to alter the transmission efficiency of the ion funnel to provide a desired ion population to the mass analyzer. Ion beam regulation by an ion funnel is shown to provide an unbiased control to within a few percent of a targeted ion intensity or abundance. The utility of ion funnel AGC was evaluated using a protein tryptic digest analyzed with liquid chromatorgraphy Fourier transform ion cyclotron resonance (LC-FTICR) mass spectrometry. The ion population in the ICR cell was accurately controlled to a variety of differenct levels, which improved data quality and provided better mass measurment accuracy.
2005. "The utility of accurate mass and LC elution time information in the analysis of complex proteomes." Journal of the American Society for Mass Spectrometry 16(8):1239-1249. Abstract Theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity and possible utility of combined peptide accurate mass and predicted LC normalized elution time (NET) information. The uniqueness of each peptide was evaluated using its combined mass (+/- 5 ppm and 1 ppm) and NET value (no constraint, +/- 0.05 and 0.01 on a 0-1 NET scale). The set of peptides both underestimates actual biological complexity due to the lack of specific modifications, and overestimates the expected complexity since many proteins will not be present in the sample or observable on the mass spectrometer because of dynamic range limitations. Once a peptide is identified from an LCMS/MS experiment, its mass and elution time is representative of a unique fingerprint for that peptide. The uniqueness of that fingerprint in comparison to that for the other peptides present is indicative of the ability to confidently identify that peptide based on accurate mass and NET measurements. These measurements can be made using HPLC coupled with high resolution MS in a high-throughput manner. Results show that for organisms with comparatively small proteomes, such as Deinococcus radiodurans, modest mass and elution time accuracies are generally adequate for peptide identifications. For more complex proteomes, increasingly accurate easurements are required. However, the majority of proteins should be uniquely identifiable by using LC-MS with mass accuracies within +/- 1 ppm and elution time easurements within +/- 0.01 NET.
2005. "Targeted Comparative Proteomics by Liquid Chromatography - Tandem Fourier ion cyclotron resonance Mass Spectrometry." Analytical Chemistry 77(2):400-406. Abstract In proteimics, effective methods are needed for identifying the relatively limited subset of proteins displaying significant changes in abundance between two samples. One way to accomplish this task is to target for identification by MD/MS only the "interesting" proteins based on the abundance ratio of isotopically labled pairs of peptides. We have developed the software and hardware tools for online LC-FTICR MS/MS studies in which a set of initially unidentified peptides from a proteome analysis can be selected for identification based on their distinctive changes in abundance following a "perturbation". We report here the validation of this method using a mixture of standard proteins combined in different ratios after isotopic labeling. We also demonstrate the application of this method to the identification of Shewanella oneidensis peptides/proteins exhibiting differential abundance in sub-oxic vs. aerobic cell cultures.
2005. "Preparation of 20-µm-i.d. Silica-based Monolithic Columns and Application for Proteomic Analysis." Analytical Chemistry 77(15):5028-5035. Abstract We report on the preparation and performance of a high efficiency 70 cm ´ 20 µm i.d. silica-based monolithic capillary column. With a mobile phase delivery pressure of 5000 psi, this monolithic column provides flow rates as low as ~40 nL/min at an LC linear velocity of ~0.24 cm/s. The resultant columns provided a separation peak capacity of ~420 under conditions of on-line coupling micro solid phase extraction (SPE) and nanoelectrospray ionization (ESI) mass spectrometry (MS) for a Shewanella oneidensis tryptic digest. A sensitivity of ~15 attomole for detection of peptides was obtained when a conventional ion trap MS/MS was used for the detection. The sensitivity and separation efficiency of this column enabled identification of 2367 different peptides from 855 S. oneidensis distinct proteins from a 2.5 µg tryptic digest sample in a single 10-h analysis by nanoLC/MS/MS. The run-to-run and column-to-column reproducibility was investigated for proteomic analyses.
2005. "Improved Proteome Coverage by Using High Efficiency Cysteinyl-peptide Enrichment: The Human Mammary Epithelial Cell Proteome." Proteomics 5(5):1263-1273. Abstract Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl-peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl-peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14,416 confidently identified peptides covering 4,294 different proteims with an estimated 10% gene coverage of the human geome. By using the high efficiency CPE, an additional 1,096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1,390 protems were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased by protein molecular weight, pI, gene location, cellular location, or bioloical functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.
2005. "Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry." Journal of Proteome Research 4(6):2070-2080. Abstract The enormous complexity, wide dynamic range of relative protein abundance of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenges the capabilities of existing analytical methodologies. We describe here the comprehensive analysis of human plasma N-glycoproteins using the combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin, enzymatically digested, and the bound, N-linked glycopeptides were released using peptide-N-glycosidase F. Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A total of 2140 different N-glycopeptides were confidently identified using stringent criteria, covering 371 non-redundant N-glycoproteins with the majority of them being extracellular or membrane proteins. The strategy significantly improved the detection, enabling the identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (~200 pg/mL), cathepsin L (~1 ng/mL), and transforming growth factor beta 1 (~2 ng/mL). A total of 712 N-glycosylation sites were identified and the confidence of these site identifications was further validated by accurate mass measurements using high resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR). This study provides the basis for future high-throughput measurements using the accurate mass and time tag approach.
2005. "Global Profiling of Shewanella oneidensis MR-1: Expression of Hypothetical Genes and Improved functional annotations." Proceedings of the National Academy of Sciences of the United States of America 102(6):2099-2104. Abstract The y-proteobacterium Shewanella oneidensis strain MR-1 is a respiratory versatile organism that can reduce a wide range of organics, metals, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the MR-1 genome were annotated as uncharacterized ‘hypothetical’ genes. We implemented an integrative approach using experimental and computational analyses to provide more detailed insight into their function. Global expression studies were conducted using RNA and protein expression profiling of cells cultivated under aerobic, suboxic, and fumaratereducing conditions, phosphate limitation and UV irradiation. transcriptomic and proteomic analyses confidently identified 538 ‘hypothetical’ genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all ‘hypothetical’ proteins). Publicly available analysis tools and databases and our own expression data were applied to improve the annotation of these genes. The annotation results were scored using a seven-category schema that ranked both confidence and precision of the functional assignment. We identified homologs for nearly all of these ‘hypothetical’ proteins (96%), thus allowing us to minimally classify them as ‘conserved proteins’. Computational and/or experimental evidence provided more precise functional assignments for 297 genes (categories 1-4; 55%). These improved functional annotations will significantly widen our understanding of vital cellular processes including signal transduction, ion transport, secondary metabolism, and transcription, as well as structural elements, such as cellular membranes. We propose that this integrative approach offers a viable means to undertake the enormous challenge of characterizing the rapidly growing number of ‘hypothetical’ proteins with each newly sequenced genome.
2005. "Utilizing human blood plasma for proteomic biomarker discovery." Journal of Proteome Research 4(4):1073-1085. doi:10.1021/pr0500657 Abstract Application of proteomic biomarker discovery efforts towards human plasma entails both incredible clinical potential as well as significant challenges to overcome the intrinsic characteristics of plasma. The dynamic range of proteins within plasma, coupled with the likely presence of potential biomarkers in the more difficult to detect lower abundance range has driven the development of various methodologies and strategies to maximize the possible detective dynamic range within this biofluid. Discussed is the array of the available approaches currently used by our laboratory and others to utilized human plasma as a viable medium for biomarker discovery efforts. Various separation, depletion, enrichment, and quantitative efforts have resulted in a measurable improvement in the detectability of the low abundance fraction of proteins but more advances are needed to bridge the gap between the current range of detection and what remains unobservable to fully maximize the potential of this sample.
2005. "Ultra-Sensitive, High Throughput and Quantitative Proteomics Measurements." International Journal of Mass Spectrometry 240(3):195-212. Abstract We describe the broad basis and application of an approach for very high throughput, ultra-sensitive, and quantitative proteomic measurements based upon the use of ultra-high performance separations and mass spectrometry. An overview of the accurate mass and time (AMT) tag approach and a description of the incorporated data analysis pipeline necessary for efficient proteomic studies are presented. Adjunct technologies, including stable-isotope labeling methodologies and improvements in the utilization of LC-MS peak intensity information for quantitative purposes are discussed. Related areas include the use of automated sample handling for improving analysis reproducibility, methods for using information from the separation for more confident peptide peak identification, and the utilization of smaller diameter capillary columns having lower volumetric flow rates to increase electrospray ionization efficiency and allow for more predictable and quantitative results. The developments are illustrated in the context of studies of complex biological systems.
2005. "Proteomic Analysis of Lyme Disease: Global Protein Comparison of Three Strains of Borrelia burgdorferi." Proteomics 5(5):1446-1453. Abstract The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric (LC-MS/MS) analysis. Protein identification was facilitated by the availability of the complete B31 genome sequence. A total of 665 Borrelia proteins were identified representing ~38 % coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.
2005. "Proteome Analysis of Liver Cells Expressing a Full- Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients." Journal of Virology 79(12):7558-7569. Abstract The development of a reproducible model system for the study of Hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full length HCV replicon. We detected > 4,400 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry (MS). The set of Huh-7.5 proteins confidently identified is, to our knowledge, the most comprehensive yet reported for a human cell line. Consistent with the literature, a comparison of Huh-7.5 cells (+) and (-) the HCV replicon identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where > 1,500 proteins were detected from 2 g protein lysate using the Huh-7.5 protein database and the accurate mass and time (AMT) tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.
2005. "Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns." Analytical Chemistry 77(18):5800-5813. Abstract Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s). Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and should be useful in algorithm development that employs improved models to predict fragment ion intensities.
2005. "Global detection and characterization of hypothetical proteins in Shewanella oneidensis MR-1 using LC-MS based proteomics based proteomics." Proteomics 5(12):3120-3130. doi:10.1002/pmic.200401140 Abstract The availability of whole genome sequences has enabled the application of powerful tools for assaying global expression patterns in environmentally relevant bacteria such as Shewanella oneidensis MR-1. A large number of genes in prokaryote genomes, including MR-1, have been annotated as hypothetical indicating that no similar protein has yet been identified in other organisms. Using high-sensitivity mass spectrometry coupled with accurate mass and time (AMT) tag methodology, 1078 tryptic peptides were collectively detected in MR-1 cultures, 671 of which were unique to their parent protein. Using only these unique tryptic peptides and a minimum of 2 peptides per protein, we identified, with high confidence, the expression of 258 hypothetical proteins. These proteins ranged from 3.5 kDa to 139 kDa, with 47 being 100 amino acid residues or less. Using a combination of information including detection in cells grown under specific culture conditions, presence within a specific cell fraction, and predictive algorithms such as PSORT and PSORT-B, possible/plausible functions are proposed for some hypothetical proteins. Further, by applying this approach a number of proteins were found not only to be expressed, but only expressed under certain culturing conditions, thereby suggesting function while at the same time isolating several proteins to distinct locales of the cell. These results demonstrate the utility of the AMT tag methodology for comprehensive profiling of the microbial proteome while confirming the expression of a large number of hypothetical genes.
2005. "Proteomics by FTICR Mass Spectrometry: Top Down and Bottom Up." Mass Spectrometry Reviews 24(2):168-200. Abstract This review offers a broad overview of recent FTICR applications and technological developments in the field of proteomics, directed to a variety of people with different expertise and interests. Both the “bottom-up” (peptide level) and “top-down” (intact protein level) approaches will be covered and various related aspects will be discussed and illustrated with examples that are among the best available references in the literature. “Bottom-up” topics include peptide fragmentation, the AMT approach and DREAMS technology, quantitative proteomics, post-translational modifications, and special FTICR software focused on peptide and protein identification. Topics in the “top-down” part include various aspects of high-mass measurements, protein tandem mass spectrometry, protein confirmations, protein-protein complexes, as well as some esoteric applications that may become more practical in the coming years. Finally, examples of integrating both approaches and medical proteomics applications using FTICR will be provided, closing with an outlook of what may be coming our way sooner than later.
2005. "Perspective: A Program to Improve Protein Biomarker Discovery for Cancer." Journal of Proteome Research 4(4):1104-1109. Abstract Biomarkers for cancer risk, early detection, prognosis, and therapeutic response promise to revolutionize cancer management. Protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology. An effective program to discover protein biomarkers using existing technology will require team science, an integrated informatics platform, identification and quantitation of candidate biomarkers in disease tissue, mouse models of disease, standardized reagents for analyzing candidate biomarkers in bodily fluids, and implementation of automation. Technology improvements for better fractionation of the proteome, selection of specific biomarkers from complex mixtures, and multiplexed assay of biomarkers would greatly enhance progress.
2005. "A Proteomic Study of the HUPO Plasma Proteome Project's Pilot Samples using an Accurate Mass and Time Tag Strategy." Proteomics 5(13):3454-3466. doi:10.1002/pmic.200401333 Abstract Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an Accurate Mass and Time (AMT) tag strategy with high-resolution mass accuracy capillary liquid chromatography Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (cLC-FTICR MS) to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published “shotgun” proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index (IPI) redundant proteins, or 377 protein families by ProteinProphet, were identified over the 6 individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/-23) as found in the serum samples (average 440+/-20). These proteins were identified by an average of 956+/-35 unique peptides in plasma and 930+/-11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput, and provided a basis for estimated quantitation.
2004. "Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes." Chapter 14 in Capillary Electrophoresis of Proteins and Peptides: Methods in Molecular Biology, vol. 276, ed. MA Strege and AL Lagu, pp. 291-304. Human Press, Totowa, NJ. Abstract Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.
2004. "The Use of Accurate Mass Tags based upon High-Throughput Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Global Proteomic Characterization." Chapter 8 in Proteome Analysis: Interpreting the Genome, ed. David W Speicher, pp. 183-224. Elsevier, Amsterdam, Netherlands. Abstract In this review, we describe the technological basis and progress towards a new global proteomics strategy that uses peptide accurate mass measurements augmented by information from separations (e.g. LC retention times) to provide large improvements in sensitivity, dynamic range, comprehensiveness and throughput. The use of ?accurate mass and time? (AMT) tags serves to eliminate the need for routine MS/MS measurements [#4109]. As the case study, we use our own research efforts to illustrate the role of AMTs within the broader context of a state-of-the-art proteomics effort. Our strategy exploits high-resolution capillary LC separations combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). AMTs represent peptide biomarkers and can be used to confidently identify proteins based on the high mass measurement accuracy provided by FTICR combined with LC elution times. Once identified using MS/MS, these biomarkers provide the foundation for subsequent high throughput studies using only AMT tags to identify and quantify the proteins expressed within a cell system. Key attractions of this approach include the feasibility of completely automated high confidence protein identifications, extensive proteome coverage, and the capability for exploiting stable-isotope labeling methods for high precision abundance measurements [#4019]. Additional developments described in this review include methods for more effective coverage of membrane proteins [#4184], for dynamic range expansion of proteome measurements [#4012], and for multi-stage separations that promise to enable more focused analyses, further extend the quality of measurements, and also extend measurements to more complex proteomes.
2004. "A rational approach for discovering and validating cancer markers in very small samples using mass spectrometry and ELISA microarrays." Disease Markers 20(3):135-148. Abstract Identifying useful markers of cancer can be problematic due to limited amounts of sample. Some samples such as nipple aspirate fluid (NAF) or early-stage tumors are inherently small. Other samples such as serum are collected in larger volumes but archives of these samples are very valuable and only small amounts of each sample may be available for a single study. Also, given the diverse nature of cancer and the inherent variability in individual protein levels, it seems likely that the best approach to screen for cancer will be to determine the profile of a battery of proteins. As a result, a major challenge in identifying protein markers of disease is the ability to screen many proteins using very small amounts of sample. In this review, we outline some technological advances in proteomics that greatly advance this capability. Specifically, we propose a strategy for identifying markers of breast cancer in NAF that utilizes mass spectrometry (MS) to simultaneously screen hundreds or thousands of proteins in each sample. The best potential markers identified by the MS analysis can then be extensively characterized using an ELISA microarray assay. Because the microarray analysis is quantitative and large numbers of samples can be efficiently analyzed, this approach offers the ability to rapidly assess a battery of selected proteins in a manner that is directly relevant to traditional clinical assays.
2004. "Proofreading activity of Pfu thermostable DNA polymerase on a 6-O-Methylguanine Containing Template Monitored by ESI-FTICR Mass Spectrometry." Chembiochem 5(7):1012-1015. Abstract Abstract DNA damage can take the form of chemical lesions that interfere with DNA polymerization and therefore, the replication of DNA within a cell. In this report we examine the effect of a particular type of base modification, a 6-O-methyl group on a guanine base. Previous reports using different DNA polymerases have identified an induced base substitution. However, this process has not been studied using polymerase chain reaction (PCR) enzyme. Electrospray ionization (ESI) mass spectrometry (MS) was used to examine the effect of this type of base on the PCR. Using Fourier transform ion cyclotron resonance (FTICR) MS, two types of amplification products were clearly resolved, one corresponding to the expected product composition, and one with a dG-dC to dA-dT base substitution. Further investigation found that the same substitution occurred when amplified with an exonuclease (exo-) form of the polymerase (lacking a proofreading function). This technique provides complementary information to other methods and is a sensitive method detecting effects of DNA damage on enzyme polymerization.
2004. "Tailored Noise Waveform/ Collision-Induced Dissociation of Ions Stored in a Linear Ion Trap Combined with Liquid Chromatography/Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." Rapid Communications in Mass Spectrometry 18(22):2682-2690. Abstract A new collision-induced dissociation (CID) technique based on broadband tailored noise waveform (TNW) excitation of ions stored in a linear ion trap has been developed. In comparison with the conventional sustained off-resonance irradiation (SORI) CID method commonly used in Fourier transform ion cyclotron resonance mass spectrometry, this MS/MS technique increases throughput by eliminating the long pump-down delay associated with gas introduction into the high vacuum ICR cell region. In addition, the TNW-CID method speeds spectrum acquisition since it does not require Fourier transformation, calculation of resonant frequencies and generation of the excitation waveforms. We demonstrate TNW-CID coupled with on-line capillary reverse phase liquid chromatography separations for identification of peptides. The experimental results are compared with data obtained using conventional quadrupole ion trap MS/MS and SORI-CID MS/MS in an ICR cell.
2004. "Identification of Proteins in Human Cytomegalovirus (HCMV) Particles: the HCMV Proteome ." Journal of Virology 78(20):10960-10966. Abstract Human cytomegalovirus (HCMV), a member of the herpesvirus family, is a large complex enveloped virus composed of both viral and cellular gene products. While the sequence of the HCMV genome has been known for over a decade, the full set of viral and cellular proteins that compose the HCMV virion are unknown. To approach this problem we have utilized gel-free two-dimensional capillary liquid chromatography-tandem mass spectrometry (MS/MS) and Fourier transform ion cyclotron resonance MS to identify and determine the relative abundances of viral and cellular proteins in purified HCMV AD169 virions and dense bodies. Analysis of the proteins from purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. In this study, we identified 71 HCMV-encoded proteins that included 12 proteins encoded by known viral open reading frames (ORFs) previously not associated with virions and 12 proteins from novel viral ORFs. Analysis of the relative abundance of HCMV proteins indicated that the predominant virion protein was the pp65 tegument protein and that gM rather than gB was the most abundant glycoprotein. We have also identified over 70 host cellular proteins in HCMV virions, which include cellular structural proteins, enzymes, and chaperones. In addition, analysis of HCMV dense bodies indicated that these viral particles are composed of 29 viral proteins with a reduced quantity of cellular proteins in comparison to HCMV virions. This study provides the first comprehensive quantitative analysis of the viral and cellular proteins that compose infectious particles of a large complex virus.
2004. "Collisional Activation of Ions in RF Ion Traps and Ion Guides: The Effective Ion Temperature Treatment." Journal of the American Society for Mass Spectrometry 15(11):1616-1628. Abstract Ion transfer and storage using inhomogeneous radio frequency (RF) electric fields in combination with gas-assisted ion cooling and focusing constitutes one of the basic techniques in mass spectrometry today. The RF motion of ions in the bath gas environment involves a large number of ion-neutral collisions that leads to the internal activation of ions. The degree of ion activation required in various applications may range from a minimum possible activation to an intense ion heating that results in ion fragmentation. Several research groups proposed using the effective temperature as a measure of ion activation under conditions of multiple ion-neutral collisions. We have developed a formalism for the effective ion temperature that accurately predicts the degree of ion activation for a given operation mode of a specific RF ion trap or ion guide. We show that RF ion activation results in near-thermal energies for ions occupying an equilibrium position at the center of an RF trap, whereas increased ion activation can be produced by shifting ions off-center, e.g., by means of an external DC electric field. The ion dissociation in the linear quadrupole ion trap using the dipolar DC ion activation has been observed experimentally and interpreted in terms of the effective ion temperature.
2004. "Charge Competition and the Linear Dynamic Range of Detection in Electrospray Ionization Mass Spectrometry." Journal of the American Society for Mass Spectrometry 15(10):1416-1423. Abstract An experimental investigation and theoretical analysis are reported on charge competition in electrospray ionization (ESI) and its effects on the linear dynamic range of ESI mass spectrometric (MS) measurements. The experiments confirmed the expected increase of MS sensitivities as the ESI flow rate decreases. However, different compounds show somewhat different mass spectral peak intensities even at the lowest flow rates, the same concentration and electrospray operating conditions. MS response for each compound solution shows good linearity at lower concentrations and levels off at high concentration, consistent with analyte “saturation” in the ESI process. The extent of charge competition leading to saturation in the ESI process is consistent with the relative magnitude of excess charge in the electrospray compared to the total number of analyte molecules in the solution. This ESI capacity model allows one to predict the sample concentration limits for charge competition and the on-set of ionization suppression effects, as well as the linear dynamic range for ESI-MS. The implications for quantitative MS analysis and possibilities for effectively extending the dynamic range of ESI measurements are discussed.
2004. "Application of Peptide LC Retention Time Information in a Discriminant Function for Peptide Identification by Tandem Mass Spectrometry." Journal of Proteome Research 3(4):760-769. Abstract We describe the application of a peptide retention time reversed phase liquid chromatography (RPLC) prediction model previously reported (Petritis et al. Anal. Chem. 99, 2002, 11049) for improved peptide identification. The model uses peptide sequence information to generate a theoretical (predicted) elution time that can be compared with the observed elution time. Using data from a set of known proteins, the retention time parameter was incorporated into a discriminant function for use with tandem mass spectrometry (MS/MS) data analyzed with the peptide/protein identification program SEQUEST. For singly charged ions, the number of identifications increased by 12% when the elution time metric is included compared to when mass spectral data is the sole source of information in the context of a Drosophila melanogaster database. A 3-4% improvement was obtained for doubly and triply charged ions for the same biological system. Application to the larger Rattus norvegicus (rat) and human proteome databases resulted in an 8-9% overall increase in the number of identifications, when both the discriminant function and elution time are used. The effect of adding “runner-up” hits (peptide matches that are not the highest scoring for a spectra) from SEQUEST is also explored, and we find that the number of confident identifications is further increased when these hits are also considered. Finally, application of the discriminant functions derived in this work with ~2.2 million spectra from 330 LC-MS/MS analyses of peptides from human plasma protein resulted in a 19% increase in confident peptide identifications (9551 vs 8049) using elution time information. Further improvements from the use of elution time information can be expected as both the experimental control of elution time reproducibility and the predictive capability are improved.
2004. "Ultra-sensitive and quantitative analyses from combined separations-mass spectrometry for the characterization of proteomes." Accounts of Chemical Research 37(4):269-278. Abstract This article describes developments in fundamental and applied aspects of separations, electrospray ionization phenomena, and mass spectrometric instrumentation that are interrelated and important for making more effective and quantitative measurements, particularly for proteomics applications. The basis for better quantitation and ultra-high sensitivity is highlighted for high resolution capillary liquid chromatography separations that provide low nL/min flow rates to an electrospray ionization interface. The increased dynamic range of measurements and sub-attomole regime detection limits obtainable open new avenues for biological research.
2004. "Understanding and Designing Field Asymmetric Waveform Ion Mobility Spectrometry Separations in Gas Mixtures." Analytical Chemistry 76(24):7366-7374. doi:10.1021/ac049299k Abstract Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) has significant potential for post-ionization separations in conjunction with MS analyses. FAIMS exploits the fact that ion mobilities in gases depend on the electric field in a manner specific to each ion, which allows one to fractionate ion mixtures. Nearly all previous work has used pure gases, for which FAIMS fundamentals are understood reasonably well. However, experiments in gas mixtures like N2/CO2 have uncovered unexpected phenomena that remained unexplained. Here we introduce a universal model for FAIMS separations in mixtures, derived from the formalisms that determine high-field mobilities in heteromolecular gases. Overall, the theoretical findings are consistent with the data in N2/CO2, though quantitative discrepancies remain. As a control, modeled results for N2/O2 fit Blanc’s law, in agreement with measurements. Calculations for He/N2 are broadly consistent with observations, and show why adding He to the working gas enhances FAIMS performance. We predict spectacular non-Blanc effects in mixtures of extremely disparate gases such as He/CO2, which could improve the peak capacity and sensitivity of technique. Understanding the FAIMS operation in gas mixtures will enable rational design of media for both targeted and global analyses.
2004. "Modeling the Resolution and Sensitivity of FAIMS analyses." Journal of the American Society for Mass Spectrometry 15(10):1487-98. Abstract Field Asymmetric waveform Ion Mobility Spectrometry (FAIMS) is rapidly gaining acceptance as a robust, versatile tool for post-ionization separations prior to mass-spectrometric analyses. The separation is based on differences between ion mobilities at high and low electric fields, and proceeds at atmospheric pressure. Two major advantages of FAIMS over condensed-phase separations are its high speed and an ion focusing effect that often improves sensitivity. While selected aspects of FAIMS performance are understood empirically, no physical model rationalizing the resolving power and sensitivity of the method and revealing their dependence on instrumental variables has existed. Here we present a first-principles computational treatment capable of simulating the FAIMS analyzer for virtually any geometry (including the known cylindrical and planar designs) and arbitrary operational parameters. The approach involves propagating an ensemble of ion trajectories through the device in real time under the influence of applied asymmetric potential, diffusional motion incorporating the high-field and anisotropic phenomena, and mutual Coulomb repulsion of ionic charges. Calculations for both resolution and sensitivity are validated by excellent agreement with representative FAIMS measurements.
2004. "Ultrasensitive Proteomics using High-Efficiency on-Line Micro-SPE-NanoLC-NanoESI MS and MS/MS." Analytical Chemistry 76(1):144-154. Abstract New approaches for ultra-sensitive proteomics are described for the characterization of complex protein (proteomic) samples of <50 ng total mass. Ultra-high sensitivity was achieved using high-efficiency 15-m i.d. capillary liquid chromatography (i.e. nanoLC) coupled on-line to a high-sensitivity Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS) through a nanoscale electrospray ionization (nanoESI) interface. The high separation efficiency (peak capacities of ~103 with average peak widths of ~15 s) and small mobile phase flow rates (~20 nL/min at optimal linear velocities of ~0.2 cm/s) from the nanoLC and the resulting high ionization efficiency of the nanoESI provided confident protein identification from <75-zeptomole of individual proteins (e.g. with 6 tryptic peptides from albumin) and an estimated ~10 zeptomole (~6000 molecules) sensitivity for peptide detection. Application of the nanoLC with ion trap MS/MS also allowed targeted protein identification at low attomole levels. The on-line coupled micro solid phase extraction allowed loading of sample solutions at 8 L/min, and provided a 250 attomolar peptide concentration detection limit using FTICR MS. This sensitivity enabled identification of proteins from 0.5 pg of a whole proteome extract tryptic digest sample. The proteome measurement dynamic range, protein identification overlap, and proteome quantitation accuracy were also investigated. An modified accurate mass and time tag data analysis methodology was used for peptide and protein identification, allowing the nanoLC-FTICR MS approach to identify 872 proteins from a 3 hour analysis of a 2.5 ng Deinococcus radiodurans proteome sample. The zeptomole level sensitivity provides a basis for extension of proteomics studies to low numbers of cells, and potentially a single mammalian cell.
2004. "Ultra-High-Efficiency Strong Cation Exchange LC/RPLC/MS/MS for High Dynamic Range Characterization of the Human Plasma Proteome." Analytical Chemistry 76(4):1134-1144. Abstract In this study, we report a comprehensive approach for ultrahigh-efficiency separations by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) for broad protein characterization of human plasma. The power of this approach is demonstrated by the confident identification of 1062 human plasma proteins based upon identification of 2992 tryptic peptides using highly conservative SEQUEST search criteria from a non-depleted human plasma sample. The approach provides a dynamic range of ~9 orders of magnitude in protein abundance using conventional ion trap MS/MS, which enabled identification of pg/mL concentration human plasma proteins (e.g. cytokines) co-existing with mg/mL-level human serum albumin. This dynamic range was obtained by combining high-efficiency reversed-phase (RP) LC coupled with efficient pre-fractionation strong cation exchange (SCX) LC to achieve ultrahigh-efficiency separations. A single-dimension, high-efficiency RPLC provided a protein identification dynamic range of 4 orders of magnitude in protein content and identified 433 human plasma proteins; while the ultrahigh-efficiency SCXLC/RPLC (i.e. 15 fractions from SCXLC), with the assistance of the SCXLC-sample component concentration (up to 102 fold), extended the protein identification dynamic range to ~9 orders of magnitude in protein content, identifying 822 human plasma proteins; combination of single- and two-dimension LC/MS/MS led to identification of 1062 human plasma proteins.
2004. "Nanoscale Proteomics." Analytical and Bioanalytical Chemistry 378(4):1037-1045. Abstract This paper describes efforts to develop a liquid chromatography (LC)/mass spectrometry (MS) technology for ultra-sensitive proteomics studies, i.e. nanoscale proteomics. The approach combines high-efficiency nano-scale LC with advanced MS, including high sensitivity and high resolution Fourier transform ion cyclotron resonance (FTICR) MS, to perform both single-stage MS and tandem MS (MS/MS) proteomic analyses. The technology developed enables large-scale protein identification from nanogram size proteomic samples and characterization of more abundant proteins from sub-picogram size complex samples. Protein identification in such studies using MS is feasible from <75 zeptomole of a protein, and the average proteome measurement throughput is >200 proteins/h and ~3 h/sample. Higher throughput (>1000 proteins/h) and more sensitive detection limits can be obtained using a “accurate mass and time” tag approach developed at our laboratory. These capabilities lay the foundation for studies from single or limited numbers of cells.
2004. "Validation of Shewanella oneidensis MR-1 Small Proteins by AMT Tag-based Proteome Analysis." OMICS. A Journal of Integrative Biology 8(3):239-254. Abstract Using stringent criteria for protein identification by accurate mass and time (AMT) tag mass spectrometric methodology, we detected 36 proteins <101 amino acids in length, including 10 that were annotated as hypothetical proteins, in 172 global tryptic digests of Shewanella oneidensis MR-1 proteins analyzed. Peptides that map to the conserved, but functionally uncharacterized proteins SO4134 and SO2787, were the most frequently detected small proteins in these samples, while hypotheticals SO2669 and SO2063, conserved hypotheticals SO0335 and SO2176, and the SlyX protein (SO1063) were observed at frequencies similar to small expected abundant ribosomal proteins and translation initiation factor IF-1 and consequently, likely to encode important cellular functions. In addition, 30 proteins including three of the small proteins that map to genes predicted to encode frameshifts, point mutations, or recoding signals were detected. Of these 30 genes, peptides that map to positions beyond internal stop codons were detected in 13 genes (SO0101, SO0419, SO0590, SO0738, SO1113, SO1211, SO3079, SO3130, SO3240, SO4231, SO4328, SO4422, and SO4657). While expression of the full-length formate dehydrogenase encoded by SO0101 can be explained by incorporation of selenocysteine at the internal stop codon, the mechanism of translating downstream sequences in the remaining genes remains unknown.
2004. "High Throughput Proteomics Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." Expert Review of Proteomics 1(1):87-95. Abstract The advent of high throughput proteomics technology for global detection and quantitation of proteins creates new opportunities and challenges for those seeking to gain greater understanding of cellular machinery. Here, we review recent advances in high-resolution capillary liquid chromatography coupled to Fourier transform ion cyclotron resonance (FTICR) mass spectrometry along with its potential application to high throughput proteomics. These technological advances combined with quantitative stable isotope labeling methodologies provide powerful tools for expanding our understanding of biology at the system-level.
2004. "Integrative Analysis of the Mitochondrial Proteome in Yeast." PloS Biology 2(6):0795-0804. Abstract In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demostrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidates genes available for mapping Mendelian and complex mitochondrial disorders in humans.
2004. "A common open representation of mass spectrometry data and its application to proteomics research." Nature Biotechnology 22(11):1459-1466. Abstract A broad range of mass spectrometers are used in mass spectrometry (MS)-based proteomics research. Each type of instrument possesses a unique design, data system and performance specifications, resulting in strengths and weaknesses for different types of experiments. Unfortunately, the native binary data formats produced by each type of mass spectrometer also differ and are usually proprietary. The diverse, nontransparent nature of the data structure complicates the integration of new instruments into preexisting infrastructure, impedes the analysis, exchange, comparison and publication of results from different experiments and laboratories, and prevents the bioinformatics community from accessing data sets required for software development. Here, we introduce the 'mzXML' format, an open, generic XML (extensible markup language) representation of MS data. We have also developed an accompanying suite of supporting programs. We expect that this format will facilitate data management, interpretation and dissemination in proteomics research.
2004. "Proteomic Analyses using an Accurate Mass and Time Tag Strategy." BioTechniques 37(4):621-636. Abstract An accurate mass and time (AMT) tag approach for proteomic analyses has been developed over the last several years to facilitate comprehensive high throughput proteomic measurements. An AMT tag database for an organism, tissue or cell line is established by initially performing standard shotgun proteomic alalysis and, most importantly, by validating peptide identifications using the mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Creation of an AMT tag database largely obviates the need for subseqent MS/MS analyses, and thus facilitates high throughput analyses. The strength of this technology resides in the ability to achieve highly efficient and reproducible 1D reversed-phased LC separations in conjunction with highly accurate mass measurements using FTICR MS. Recent improvements allow analysis of as little as picrogram amounts of proteome samples by minimizing sample handling and maximizing peptide recovery. Nanoproteomics platform has also demonstrated the ability to detect over 10 9 differences in protein abundance in human plasma and identify more abundant proteins from sub-picogram amounts of samples. The AMT tag approach is poised to become a new standard technique for the in-depth and high throughput analysis of complex organisms, clinical samples, with potential to extend the analysis to single mammalian cell.
2004. "FTICR Mass Spectrometry for Qualitative and Quantitative Bioanalyses." Current Opinion in Biotechnology 15(1):3-11. Abstract Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is playing an increasing role in the characterization of cellular systems due to its capabilities for providing higher confidence of identification, increased dynamic range, and sensitivity unmatched by other MS platforms. Particularly in Proteomics, where global and quantitative approaches are essential, the attributes of FTICR-MS are poised to make significant contributions. This review covers the recent advances in the field with an emphasis on proteomic applications. It includes high performance microcolumn separation techniques coupled to FTICR, as well as methods that improve protein identification, sensitivity, dynamic range, and throughput.
2004. "High throughput comparative proteome analysis using a quantitative cysteinyl-peptide enrichment technology." Analytical Chemistry 76(18):5345-5353. Abstract A new quantitative cysteinyl-peptide enrichment technology (QCET) was developed to achieve higher efficiency, greater dynamic range, and higher throughput in quantitative proteomics that use stable-isotope labeling techniques combined with high resolution liquid chromatography (LC)-mass spectrometry (MS). This approach involves 18O labeling of tryptic peptides, high efficiency enrichment of cysteine-containing peptides, and confident protein identification and quantification using the accurate mass and time tag strategy. Proteome profiling of naïve and in vitro-differentiated human mammary epithelial cells using QCET resulted in the identification and quantification of 603 proteins in a single LC-Fourier transform ion cyclotron resonance MS analysis. Advantages of this technology include: (1) a simple, highly efficient method for enriching cysteinyl-peptides; (2) a high throughput strategy suitable for extensive proteome analysis; and (3) improved labeling efficiency for better quantitative measurements. This technology enhances both the functional analysis of biological systems and the detection of potential clinical biomarkers.
2004. "Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry." Journal of Mass Spectrometry 39(2):206-215. Abstract In contrast to lower phosphorylation states (e.g., the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine -casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine -casein) has often been problematic for liquid chromatography-mass spectrometry analysis due to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides upon addition of phosphoric acid (0.1% to 1.0%) to the sample solution; a 10-fold increase in sensitivity was measured for the detection of two tryptic phosphopeptides as well as a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with an ion trap tandem mass spectrometer for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C-18-bonded silica surface.
2004. "Multidimensional Proteome Analysis of Human Mammary Epithelial Cells." Journal of Proteome Research 3(1):68-75. Abstract Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with strong cation exchange chromatography to detect tryptically digested peptides in the global protein mixture of human mammary epithelial cells (HMECs) based upon the use of very high resolution, reversed phase capillary LC–MS/MS. A total of >6,200 unique peptides were identified with high confidence covering 1,700 different proteins, out of which 93% were mapped to chromosomal locations providing an estimated 4.4% coverage of the annotated human genome based upon the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.
2004. "Dissociation Behavior of Doubly-Charged Tryptic Peptides: Correlation of Gas-Phase Cleavage Abundance with Ramachandran Plots." Journal of the American Chemical Society 126(10):3034-3035. Abstract Large numbers of gas-phase dissociation spectra of protonated peptides are obtained daily and used in protein identification studies. Yet fundamental knowledge of the factors that influence their unimolecular dissociation branching ratios is relatively poor. It is still not possible to predict dissociation branching ratios from peptide sequence. Clearly, several chemicals factors must influence dissociation patterns, includes y, f angles determined by the residues involved in an amide bond, the propensities for certain side chains to interact each other or with the backbone, the tendency for added protons to be intramolecularly solvated, and the stability of the fragment ions once formed.
2004. "Consideration for Electron Capture Dissociation Efficiency in FTICR Mass Spectrometry." International Journal of Mass Spectrometry 234(1-3):131-136. Abstract An experimental approach for increasing efficiency of Electron Capture Dissociation (ECD) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) is presented. This approach is based on manipulation of the spatial distribution of ion cloud inside an FTICR trap during electron irradiation, which is realized using both on-resonance pre-excitation of the ions and sustained off-resonance irradiation (SORI). Fragmentation efficiency achieved is compared with theoretical prediction. The method may be useful in biological applications of FTICR where ECD technique is most applicable, such as identification of posttranslational modifications in proteins and de novo sequencing.
2004. "Global Analysis of the Membrane Subproteome of Pseudomonas aeruginosa using Liquid Chromatography-Tandem Mass Spectrometry." Journal of Proteome Research 3(3):434-444. Abstract Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants or cancer. Liquid chromatography coupled online with tandem mass spectrometry (LC-MS/MS) was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (TMD; ranging from 1 to 14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents the most comprehensive qualitative proteomic analysis of the membrane subproteome of P. aeruginosa and for prokaryotes in general to date.
2004. "An Automated High Performance Capillary Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometer for High-Throughput Proteomics." Journal of the American Society for Mass Spectrometry 15(2):212-232. Abstract We report on a fully automated 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer coupled to reverse-phase chromatography for high-throughput proteomic studies. Modifications made to the front-end of a commercial FTICR instrument – a dual-ESI-emitter ion source; dual-channel electrodynamic ion funnel; and collisional-cooling, selection and accumulation quadrupoles – significantly improved the sensitivity, dynamic range and mass measurement accuracy of the mass spectrometer. A high-pressure capillary liquid chromatography (LC) system was incorporated with an autosampler that enabled 24 h/day operation. A novel method for accumulating ions in the ICR cell was also developed. Unattended operation of the instrument revealed the exceptional reproducibility (1-5% deviation in elution times for peptides from a bacterial proteome), repeatability (10-20% deviation in detected abundances for peptides from the same aliquot analyzed a few weeks apart) and robustness (high-throughput operation for 5 months without downtime) of the LC/FTICR system. When combined with modulated-ion-energy gated trapping, the internal calibration of FTICR mass spectra decreased dispersion of mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations to < 5 ppm over a dynamic range for each spectrum of 10 3.
2003. "Proteome Analysis by Mass Spectrometry." In Annual Review in Biophysics and Biomolecular Structure, vol. 32, ed. Robert M. Stroud, pp. 399-424. Annual Reviews, Palo Alto, CA. Abstract The coupling of high-performance mass spectrometry instrumentation with highly efficient chromatographic and electrophoretic separations has enabled rapid qualitative and quantitative analysis of thousands of proteins from minute samples of biological materials. Here, we review recent progress in the development and application of mass spectrometry-based techniques for the qualitative and quantitative analysis of global proteome samples. Techniques such as multidimensional peptide and protein separations coupled with mass spectrometry, accurate mass measurement of peptides from global proteome digests, and mass spectrometric characterization of intact proteins hold great promise for characterization of highly complex protein mixtures. Advances in chemical tagging and isotope labeling techniques have enabled quantitative analysis of proteomes, and highly specific isolation strategies have been developed and aimed at selected analysis of post-translationally modified proteins.
2003. "An extremely stable, self-complementary hydrogen-bonded duplex ." Chemical Communications 13:1556-1557. Abstract This paper describes the design, synthesis and characterization of a self-complementary six-H-bonded duplex with an association constant greater than 10^9/M in CHCl3. Numerous unnatural self-assembly systems have been developed in recent years. Most of these previously described systems are case-dependent, i.e., the individual components carry the information that defines only the formation of the specific assembly. An alternative approach involves the design of highly specific and highly stable recognition units (modules)that are compatible with a variety of structural components. Such recognition modules or �molecular glues" then direct the assembly of these structural components. In this regard,hydrogen-bonded complexes based on rigid heterocycles with multiple H-bonding donor (D) and acceptor (A) sites have received the most attention in recent years. Other complexes, most based on H-bonding interactions, have also been reported. Highly stable, self- complementary H-bonded complexes are particularly attractive for developing supramolecular homopolymers of very high molecular weights. In spite of the intriguing perspective, only a very small number of self-complementary H-bonded complexes with high stabilities are known. The best known examples involve two pairs of quadruply H-bonded, self-complementary complexes, both based on the AADD-DDAA array, and with association constants greater than 10^7/M. We report here the design and characterization of our first six-H-bonded, self-complementary duplex that contains the AADADD-DDADAA array.
2003. "Duplex Foldamers from Assembly Induced Folding." Journal of the American Chemical Society 125(33):9932-9933. Abstract .
2003. "Proteomic characterization of nipple aspirate fluid: Identification of potential biomarkers of breast cancer." Breast Cancer Research and Treatment 80(1):87-97. doi:10.1023/A:1024479106887 Abstract Mammary ductal cells are the origin for 70 to 80% of breast cancers. Nipple aspirate fluid (NAF) contains proteins directly secreted by the ductal and lobular epithelium in non-lactating women. Proteomic approaches offer a largely unbiased way to evaluate NAF as a source of biomarkers and are sufficiently sensitive for analysis of small NAF volumes (10 to 50 ml). In this study, we initially evaluated a new process for obtaining NAF and discovered that this process resulted in a volume of NAF that was suitable for analysis in ~90% of subjects. Proteomic characterization of NAF identified 64 proteins. Although this list primarily includes abundant and moderately abundant NAF proteins, very few of these proteins have previously been reported in NAF. At least 15 of the NAF proteins identified have previously been reported to be altered in serum or tumor tissue from women with breast cancer, including cathepsin D and osteopontin. In summary, this study provides the first characterization of the NAF proteome and identifies several candidate proteins for future studies on breast cancer markers in NAF.
2003. "Proteomic Characterization of Nipple Aspirate Fluid: Identification of Potential Biomarkers of Breast Cancer." Breast Cancer Research and Treatment 80(1):87-97. Abstract Mammary ductal cells are the cellular origin for 70 to 80% of breast cancers. Nipple aspirate fluid (NAF) contains proteins directly secreted by the ductal and lobular epithelium in non-lactating women. Proteomic approaches offer a largely unbiased way to evaluate NAF as a source of biomarkers and are sufficiently sensitive for analysis of small NAF volumes (10 to 50 ml). In this study, we initially evaluated a new process for obtaining NAF and discovered that this process resulted in adequate volume of NAF suitable for analysis in ~90% of subjects. Proteomic characterization of NAF identified 64 proteins. Although this list primarily includes abundant and moderately abundant NAF proteins, very few of these proteins have previously been reported in NAF. At least 15 of the NAF proteins identified have previously been reported to be altered in serum or tumor tissue from women with breast cancer, including cathepsin D and osteopontin. In summary, this study provides the first characterization of the NAF proteome and identifies several candidate proteins for future studies on breast cancer markers in NAF.
2003. "Proteomic Characterization of Nipple Aspirate Fluid: Identification of Potential Biomarkers of Breast Cancer ." Breast Cancer Research and Treatment 80(1):87-97. Abstract Mammary ductal cells are the cellular origin for 70 to 80% of breast cancers. Nipple aspirate fluid (NAF) contains proteins directly secreted by the ductal cells in non-lactating women. Proteomic approaches offer a largely unbiased way to evaluate NAF as a source of biomarkers and are sufficiently sensitive for analysis of small NAF volumes (10 to 50 ml). In this study, we initially evaluated a new process for obtaining NAF and found that this process resulted in volumes of NAF suitable for analysis in ~90% of subjects. Proteomic characterization of NAF identified 64 proteins. Although it is likely this list primarily includes abundant and moderately abundant NAF proteins, very few of these proteins have previously been reported in NAF. At least 15 of the NAF proteins identified have previously been reported to be altered in serum or tumor tissue from women with breast cancer, including cathepsin D and osteopontin. In summary, this study provides the first characterization of the NAF proteome and identifies several candidate proteins for future studies on breast cancer markers in NAF.
2003. "Suppression of the Lower Charge State Ions in the External Accumulation RF Multipole with a Reduced Trapping DC Potential." Journal of the American Society for Mass Spectrometry 14(11):1229-1235. Abstract Radio frequency (RF) multipoles are increasingly used in mass spectrometry as two-dimensional ion traps for ion accumulation and pre-selection. It was reported recently that ions having lower charge states, in particular singly charged ions, can be efficiently removed from such an ion trap when reduced DC trapping voltages are applied. The procedure can be very useful in removing singly charged species contributing chemical noise to mass spectra of complex bio-molecular samples, e.g. solvent contaminants in LC-MS or ampholytes in CIEF-MS experiments. We consider a physical mechanism and derive relationships that provide a quantitative description for the process of low charge state ejection. Experimental conditions for the efficient discrimination against lower charge states are evaluated. Initial experimental observations reported are in agreement with the theoretical treatment.
2003. "Modeling the ion density distribution in collisional cooling RF multipole ion guides." International Journal of Mass Spectrometry 222(1-3):155-174. Abstract Collisional cooling radio frequency (RF) multipoles are widely used in mass spectrometry, as ion guides and two-dimensional (2D) ion traps. Understanding the behavior of ions in these devices is important in choosing a multipole configuration. We have developed a computer model based on ion trajectory calculations in the RF multipole electric field, taking into account ion-ion and ion-neutral interactions. The two-dimensional model for idealized infinite RF multipoles gives accurate description of the ion density distribution. We consider first a basic case of a single m/z ion cloud in the 2D RF quadrupole after equilibrium is reached. Approximate theoretical relationships for the ion cloud configuration in the 2D ion trap are tested based on simulations results. Next we proceed with a case of an ion cloud consisting of several different m/z ion species. The ion relaxation dynamics and the process of establishing the stratified ion density distribution are followed. Simulations reveal a different relaxation dynamics for the axial and radial ion kinetic energy components. The kinetic energy relaxation rate is dependent on ion population and bath gas pressure. The equilibrium distribution agrees well with the ion stratification theory, as demonstrated by simulations for RF quadrupole and octopole 2D ion traps.
2003. "Proteome Analyses Using Accurate Mass and Elution Time Peptide Tags with Capillary LC Time-of-Flight Mass Spectrometry." Journal of the American Society for Mass Spectrometry 14(9):980-991. Abstract We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al. Proc. Natl Acad. Sci. USA, 99, 2002, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are logged in a accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e. using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; ~3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, ~900 ORFs detected using LC-TOFMS, with ~500 ORFs covered by at least two AMT tags. These results indicate that AMT databases searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 hours. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized.
2003. "High Mass Measurement Accuracy Determination for Proteomics using Multivariate Regression Fitting: Application to Electrospray Ionization Time-Of-Flight Mass Spectrometry." Analytical Chemistry 75(3):460-468. Abstract Abstract. Important factors that limit the mass measurement accuracy from a mass spectrometer are related to (1) the type of mass analyzer used and (2) the data processing/calibration methods used to obtain mass values from the raw data. Here, two data processing methods are presented that correct for systematic deviations when measuring the mass of ions using a time-of-flight (TOF) mass spectrometer. The first fitting method is one where m/z values are obtained from fitting peak distributions using double Gaussian functions. A second calibration method takes into account the slight non-linear response of the time-of-flight analyzer in addition to the drift in the calibration over time. Using multivariate regression, both of these two effects can be corrected for using a single calibration formula. Achievable performance was evaluated with a trypsin digestion of serum albumin and proteins from the organism D. radiodurans that were analyzed using gradient reverse-phase liquid chromatography combined with electrospray ionization orthogonal TOF mass spectrometer. The root mean square deviation between the theoretical and experimental m/z for serum albumin was found to be 8 ppm using the double Gaussian-multivariate method compared to 29 ppm determined using linear calibration and normal peak centroiding. An advantage of the methods presented here is that no calibrant compounds need to be added to the mobile phase, thereby avoiding interference effects and signal suppression of analytes.
2003. "High-Efficiency On-Line Solid-Phase Extraction Coupling to 15-150 um I.D. Column Liquid Chromatography for Proteomic Analysis." Analytical Chemistry 75(14):3596-3605. Abstract Flexible manipulation of various properties of proteomic samples is important for proteomic analyses, but it has been little explored for newly developed approaches based on liquid chromatography (LC) in combination with mass spectrometry (MS). With miniaturization of the LC column inner diameter dimensions (required for improving the analysis sensitivity), this issue becomes more challenging due to the small flow rates and the increasing effects of extra column volume on the separation quality and its use for resolving complex proteomic mixtures. In this study, we used commercial switching valves (150-mm channels) to implement the on-line coupling of capillary LC columns with relatively large solid phase extraction (SPE) columns operated at 10,000 psi. With optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained with peak capacities of ~1000 for capillaries having inner diameters between 15 to 150 mm. The on-line coupled SPE columns increased the sample processing capabilities by ~400-fold for sample solution volume and ~10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Used with an ion trap tandem MS we could typically identify 1100-1500 peptides for analyses in a single 5-hour run. Peptides extracted on the SPE column and eluted from the LC column covered a hydrophilicity/hydrophobicity range that include an estimated ~98% of all the tryptic peptides. The present implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for routine proteomic analyses.
2003. "Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures." Analytical Chemistry 75(20):5441-5450. Abstract Many cellular processes are regulated by reversible protein phosphorylation and the ability to identify and quantify phosphoproteins from proteomes is essential for gaining a better understanding of these dynamic cellular processes. However, a sensitive, efficient and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) for isolating and measuring the relative abundance of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported Phosphoprotein Isotope-coded Affinity Tag (PhIAT)approach developed by our laboratory1-2, where the O-phosphate moiety on phosphoseryl or phosphothreonyl residues were derivatized by hydroxide ion-medated B-elimination followed by the addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either ligh (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary LC-MS/MS. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures was demonstrated using casein phosphoproteins. Its utility for proteomic applications is demonstrated by the labeling of soluble proteins from human breast cancer cell line.
2003. "Use of artificial neural networks for the accurate prediction of peptide liquid chromatography elution times in proteome analyses." Analytical Chemistry 75(5):1039-1048. Abstract The use of artificial neural networks (ANNs) is described for predicting the reversed-phase liquid chromatography retention times of peptides enzymatically digested from proteome-wide proteins. In order to enable the comparison of the numerous LC-MS data sets a genetic algorithm was developed to normalize the peptide retention data into a range (from 0 to 1), improving the peptide elution time reproducibility to about 1%. The network developed in this study was based on amino acid residue composition and consists of 20 input nodes, 2 hidden nodes and 1 output node. A data set of about 7000 confidently identified peptides from the microorganism Deinococcus radiodurans was used for the training of the ANN. The ANN was then used to predict the elution times for another set of 5200 peptides tentatively identified by MS/MS from a different microorganism (Shewanella oneidensis). The model was found to predict the peptides of elution time with up to 54 amino acid residues (the longest peptide identified after tryptic hydrolysis of S. oneidensis) with an average accuracy of 3%. This predictive capability was then used to distinguish with high confidence isobar peptides otherwise indistinguishable by accurate mass measurements as well as to uncover peptide misidentifications. Thus, integration of ANN peptide elution time prediction in the proteomic research will increase both the number of protein identifications and their confidence.
2003. "Integration of Electrokinetic-Based Multidimensional Separations/Concentration Platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for Proteome Analysis of Shewanella Oneidensis." Analytical Chemistry 75(17):4432-4440. Abstract .
2003. "Identification of Tryptic Peptides from Large Databases using Multiplexed Tandem Mass Spectrometry: Simulations and Experimental Results." Proteomics 3(7):1279-1286. Abstract Multiplexed MS/MS was recently demonstrated as a means to increase the throughput of peptides identification in LC-MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteome database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to 10 peptides from a single dataset with only 3 “y” or “b” fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC-multiplexed FTICR MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.
2003. "Stable Isotope-Coded Proteomic Mass Spectrometry." Current Opinion in Biotechnology 14(1):101-109. Abstract The ability to quantify the changes in protein abundance between cells subjected to a variety of extracellular stimuli or the onset of a diseased state remains an extremely active area of proteome research. Although advances in sample preparation, chromatographic separation, mass spectrometry instrumentation and bioinformatics contribute to producing a viable method for comparative proteome-wide analyses, the foundation of quantitation is based in part upon improved methods for chemical and metabolic stable isotope labeling of proteins and peptides. The ability to quantify differences in protein expression and post-translational modifications has been demonstrated, but insights into the biochemical mechanisms that will contribute to the development of new biotechnologies have yet to be realized.
2003. "Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis." Journal of Proteome Research 2(2):153-161. Abstract The ability to identify and quantify integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and derivatize these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified of which 40 were integral membrane proteins containing from 1 to 9 mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g. ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g. pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.
2003. "Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells." Proteomics 3(8):1647-1651. Abstract The cell membrane serves as the primary interface of the cell with its surrounding environment. Events involving cell-cell communication, binding of chemical messengers, interaction with pathogens, environmental perturbations, transport functions, etc. are often mediated by specific interactions with cell surface-exposed membrane proteins. Cellular differentiation and other highly regulated parallel biochemical processes determine the protein composition of the cell membrane, and the dynamic turnover of membrane proteins provides a unique opportunity for the application of new proteomic approaches. Information obtained from methods investigating cell membrane subproteomes could potentially reveal specific target sites that are useful for diagnostics and ultimately for application of pharmaceutical therapeutics.
2003. "Initial Implementation of external accumulation liquid chromatography/electrospray ionization Fourier transform ion cyclotron resonance with automated gain control." Rapid Communications in Mass Spectrometry 17(7):627-636. Abstract Capillary LC separation coupled with external accumulation FTICR mass spectrometry has recently been demonstrated to have significant potential for proteomics research. Accumulation of an excessive space charge in an external to the FTICR cell ion trap has been shown to result in increased mass measurement error, undesirable ion discrimination and/or fragmentation, and potentially causing misrepresentation or incorrect assignments of lower abundance peptides in the acquired mass spectra. In this work we report on the capability of data-dependent adjustment of ion accumulation times in the course of LC separations, further referred to as Automated Gain Control (AGC). Three different AGC approaches were evaluated based upon the number of putative peptides from a tryptic digest of four casein proteins detected in the course of LC/FTICR separations. AGC was found to increase by up to a factor of 3 the number of peptides identified compared to the conventional technique.
2003. "Automated Gain Control and Internal Calibration With External Ion Accumulation Capillary liquid chromatography-electrospray ionization-fourier transform ion cyclotron resonance." Analytical Chemistry 75(16):4195-4205. Abstract When combined with capillary LC separations, Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FTICR MS) has increasingly been applied for advanced characterization of proteolytic digests. Incorporation of external (to the ICR cell) ion accumulation multipoles with FTICR for ion pre selection and accumulation has enhanced the dynamic range, sensitivity and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in “overfilling” of the external trap, resulting in m/z discrimination and fragmentation of peptide ions. An excessive space charge trapped in the ICR cell causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) for protein identification. To eliminate m/z discrimination in the external ion trap, further increase the duty cycle and improve MMA, we developed a capability for data-dependent adjustment of ion accumulation times in the course of an LC separation, referred to as Automated Gain Control (AGC), in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The system was initially evaluated in the analysis of a 0.5 mg/mL tryptic digest of bovine serum albumin. The implementation of LC/ESI/AGC/FTICR with internal calibration gave rise to a ~10-fold increase in the number of identified tryptic peptides within mass measurement accuracy of 2 ppm as compared to that detected during the conventional LC/FTICR run with a fixed ion accumulation time and external calibration.
2002. "Advanced Mass Spectrometric Approaches for Rapid and Quantitative Proteomics." Chapter 8 in Applied Electrospray Mass Spectrometry, ed. BN Pramanik,, AK Ganguly & ML Gross, pp. 307-360. Marcel Dekker, New York, NY. Abstract With the completion of several dozen genome sequences and the first draft of the human genome in 2000, biological research is moving rapidly into the "post-genomic era". Contributing to the movement towards this era are recent advances in robotics, DNA sequencing technology, and computational analysis, all of which are resulting in an increasingly large amount of DNA sequence data, along with an array of experimental and bioinformatic tools increasingly being used for its analysis. In the post-genomic era, studies will be designed to characterize complex cellular "systems", consisting of networks of molecular networks. In the new paradigm, cellular processes are increasingly subject to global study and modeled from the "top-down", leading to new understandings of the cellular functions of the individual system constituents, how they respond to environmental perturbations, and the emergent properties arising from the complex nature of their interactions. A major goal is to understand both the molecular and cellular processes provides a basis for understanding the robustness of cellular systems, the possible modular nature of the cellular machinery, the nature of epigenetic and multigenic diseases, individual variability is susceptibility to disease, and for developing predictive capabilities of the effects arising from external perturbations. Additionally, the information gained potentially leads to an understanding of the molecular "nodes" that can be targeted for drug development, gene therapy, genetic manipulations, etc.
2002. "Laser-Machined Microdevices for Mass Spectrometry." Chapter 6 in Mass Spectrometry and Hyphenated Techniques in Neuropeptide Research, ed. J. Silberring and R. Ekman, pp. 171-192. John Wiley & Sons, Wiley Interscience, NY. Abstract In this book chapter we summarize the most recent work conducted in our lab on the development of laser-machined plastic microfluidic devices for rapid and automatic sample cleanup.
2002. "Proteome Analysis of Camptothecin-Treated Cortical Neurons using Isotope-Coded Affinity Tags." Electrophoresis 23:1591-1598. Abstract .
2002. "Frequency Shifts Due to the Interference of Resolved Peaks in Magnitude-Mode Fourier-Transform Ion Cyclotron Resonance Mass Spectra." Journal of the American Society for Mass Spectrometry 13(4):387-401. Abstract N/A
2002. "Independent Control of Ion Transmission in a Jet Disrupter Dual-Channel Ion Funnel Electrospray Ionization MS Interface." Analytical Chemistry 74(20):5431-5437. Abstract While the use of an internal calibrant can significantly increase mass measurement accuracy in many LC-MS applications, ion suppression in the ESI process and space charge effects in the MS analyzer often limit its effectiveness. These limitations are especially problematic when LC-MS is used for analyzing complex biological samples where both high sensitivity and broad dynamic range are required. To address this need, a new ESI interface has been developed to control ion transmission through the first vacuum stage of the mass spectrometer. The initial design using a dual electrospray, a dual heated capillary and a dual inlet ion funnel (Fig. 1) is specifically aimed at providing a method for controlled introduction of calibrant ions in highly accurate mass measurements using Fourier transform ion cyclotron resonance mass spectrometer.
2002. "Trends in mass spectrometry instrumentation for proteomics." Trends in Biotechnology 20(12):S3-S7. Abstract Mass spectrometry has become a primary tool for proteomics due to its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the needs for increased capabilities for proteome measurements are immense and are now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements, and promise more than order of magnitude improvements in sensitivity, dynamic range, and throughput for proteomic analyses in the near future.
2002. "The Use of Accurate Mass Tags for High-Throughput Microbial Proteomics." OMICS. A Journal of Integrative Biology 6(1):61-90. Abstract We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide accurate mass tags (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from potential mass tags identified using conventional tandem mass spec-trometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and ac-curate FT-ICR measurements has been shown capable of characterizing peptide mix-tures of significantly more than 10 5 components with mass accuracies of _1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian pro-teomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for _60% of the potentially expressed proteins in the organism Deinococcus radiodurans.
2002. "An Accurate Mass Tag Strategy for Quantitative and High-Throughput Proteome Measurements." Proteomics 2:513-523. Abstract We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness and throughput of proteomic measurements based upon the use of polypeptide "accurate mass tags" (AMTs) produced by a global protein enzymatic digestion. The two stage strategy exploits Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to first validate polypeptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing the basis for subsequent measurements without the need for MS/MS. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of significantly more than 105 components with mass accuracies of <1 ppm, sufficient for broad protein identification using AMTs. Attractions of the approach include the capability for automated high confidence protein identification, broad and unbiased proteome coverage, the capability for exploiting stable-isotope labeling methods to realize high precision for relative protein abundance measurements, and the potential for study of mammalian proteomes when combined with additional sample fractionation. The strategy is demonstrated by selected examples using Saccharomyces cerevisiae, Deinococcus radiodurans, and mouse melanoma cells.
2002. "Advanced Mass Spectrometric Methods for the Rapid and Quantitative Characterization of Proteomes." Journal of Structural and Functional Genomics 3(2):143-150. Abstract N/A
2002. "Proteomics Based on High-Efficiency Capillary Separations." Electrophoresis 23:3106-3124. Abstract .
2002. "High-Efficiency Nanoscale Liquid Chromatography Coupled On-Line with Mass Spectrometry using Nanoelectrospray Ionization for Proteomics." Analytical Chemistry 74:4235-4249. Abstract .
2002. "Myxoma Virus Immunomodulatory Protein M156R is a Structural Mimic of Eukaryotic Translation Initiation Factor eIF2 alpha." Journal of Molecular Biology 322(5):943-954. Abstract M156R, the product of the myxoma virus M156R open reading frame, is a protein of unknown function. However, several homologs of M156R from other viruses are immunomodulatory proteins that bind to interferon-induced protein kinase PKR and inhibit phosphorylation of the eukaryotic translation initiation factor eIF2a. In this study, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel b-barrel with two of the strands connected by a long loop and a short a-helix. The similarity between M156R and the predicted S1 motif structure of eIF2a suggests that the viral homologs are pseudosubstrate inhibitors of PKR that mimic eIF2a in order to compete for binding to PKR. A homology modeled structure of the well studied vaccinia virus K3L was generated based on alignment with M156R. Residues important for binding to PKR are conserved residues on the surface of the b-barrel and in the mobile loop, identifying the putative PKR recognition motif.
2002. "Increased Proteome Coverage for Quantitative Peptide Abundance Measurements Based upon High Performance Separations and DREAMS FTICR Mass Spectrometry." Journal of the American Society for Mass Spectrometry 13(8):954-963. Abstract There is no abstract currently available for this item
2002. "Gene expression profiling using advanced mass spectrometric approaches." Journal of Mass Spectrometry 37(12):1185-1198. Abstract In the era of systems biology, computational and high-throughput experimental biological approaches are increasingly being combined to provide global snapshots of entire genomes and proteomes under tissue- and disease-specific conditions. The aim is to identify proteins changing in concentration and/or post-translational state and/or location, and develop a better molecular level understanding of the operation of biological systems. Here we describe an approach for comparative proteomics that builds upon the combination of high-performance nano-scale separations with the high-mass measurement accuracy, mass-resolving power and sensitivity of Fourier transform ion cyclotron resonance mass spectrometry to provide broad dynamic range, comprehensive and quantitiative proteome measurements.
2002. "Mass Measurement Errors Caused by "Local" Frequency Perturbations in FTICR Mass Spectrometry"." Journal of the American Society for Mass Spectrometry 13:99-106. Abstract N/A
2002. "Selective Incorporation Acids for Identification Proteome-wide Level." Journal of Mass Spectrometry 37(1):99-107. Abstract The post genomic era and increased demands for broad proteome measurements and have greatly increased the needs for protein identification. We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae. Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D10) had been added. The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum. The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein. Proteins can then often be unambiguously identified based on their molecular mass and the additional constraint provided by number of Leu residues. The identities of proteins were further confirmed by repeating CIEF-FTICR measurements with samples that contain other isotopically labeled amino acid residues. Theoretical study on the amino acid composition (for a difference of the amino acid sequence) showed how many constrains are needed to unambiguously identify the protein. Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications.
2002. "Charge Effects for Differentiation of Oligodeoxynucleotide Isomers Containing 8-oxo-dG Residues." Journal of the American Society for Mass Spectrometry 13:195-199. Abstract Dissociation reactions of a series of multiply charged oligonucleotides anions were studied using an ion trap mass spectrometer. These mixed-nucleobase 12-mers fragment first by loss of a nucleobase (A, G, C and/or 5-methyl-cytosine) followed by cleavage at 3' C-O bond of the sugar from which the base is lost to produce the complementary sequence ions, i.e. a-B and w type of ions. No detectable loss of 8-oxo-guanine and/or thymine from these 12-mers is observed for the gentle collision conditions in the ion trap. The primary loss of a nucleobase and the subsequent backbone cleavage to generate sequence ions strongly depend on the charge state of the parent molecular ion. For low charge states (-2 and ?3), product ions due to the loss of a neutral guanine base and related sequence ions are dominant in the tandem mass spectra. However, preferential loss of a neutral adenine becomes the primary reaction channel from the ?5 charge state of the molecular ion. Such charge state dependent fragmentation behavior was utilized to determine the sites of 8-oxo-dG residue in a series of structural isomers. The position of 8-oxo-dG residue can be simply determined from the fragmentation pattern of ?3 charge state, but not of ?5 charge state. The strategy illustrated here for positional mapping of damaged residues in oligonucleotides is highly sensitive due to effective dynamic range enhancement in the product ion spectra by accessing the sequence informative reaction channels.
2002. "Global Analysis of Deinococcus Radiodurans Proteome by Csing Accurate Mass Tags." Proceedings of the National Academy of Sciences of the United States of America 99(17):11049-11054. Abstract The ability to understand biological systems and their constituents would be greatly facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes e.g. in response to environmental perturbations. To this end we have developed new instrumentation and a high throughput methodology to characterize an organism's dynamic proteome based upon the combination of global enzymatic digestion, high-resolution liquid chromatographic separations and analysis by Fourier transform ion cyclotron resonance mass spectrometry. Using accurate mass tags, 61% of the predicted proteome of the ionizing radiation resistant bacterium Deinococcus radiodurans was characterized with high confidence. This represents the broadest proteome coverage for any organism to date, and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.
2002. "Direct Mass Spectrometric Analysis of Intact Proteins of the Yeast Large Ribosomal Subunit using Capillary LC/FTICR." Proceedings of the National Academy of Sciences of the United States of America 99(9):5942-5947. Abstract Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry coupled with capillary reverse phase LC (RPLC) was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accurancy, achieved by "mass locking" with an internal standard froma dual ESI source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on co-translational and post-translational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.
2002. "Evaluation of Enzymatic Digestion and Liquid Chromatography-Mass Spectrometry Peptide Mapping of the Integral Membrane Protein Bacteriorhodopsin." Electrophoresis 23(18):3224-3232. Abstract .
2002. "ESI-FTICR Mass Spectrometry Employing Data-Dependent External Ion Selection and Accumulation." Journal of the American Society for Mass Spectrometry 13(2):144-154. Abstract N/A
2002. "Phosphoprotein Isotope-coded Affinity Tags: Application to the Enrichment and Identification of Low-Abundance Phosphoproteins." Analytical Chemistry 74(3):607-616. Abstract A novel approach using different isotopic labeling and biotinylation has been developed for the enrichment and quantitation of phosphoseryl and phosphothreonyl-peptides. The phosphoprotein isotope-coded affinity tag (PhIAT) exploits the high affinity biotin-avidin interaction to isolate modified phosphopeptides from a complex mixture of peptides. The PhIAT strategy for quantifying and enriching mixtures for phosphopeptides was demonstrated using a commercially available sample of the phosphoprotein B-casein. A denatured solution of B-casein was labeled using the PhIAT method and after proteolytic digestion, the labeled peptides were isolated using immobilize avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from B-casein were identified as were phophorylated peptides from as1-casein and ase-casein, known low-level (<5%) contaminants of commercially available B-casein. All of the identified phosphopeptides from these caseins have been previously documented to be phosphorylated at the sites elucidated by the PhIAT approach. The results illustrate the efficancy of the PhIAT-labeling strategy to enrich mixtures for phosphopeptides and permit the detection and identification of low abundance phosphopeptides. In addition, experiments using light and heavy isotopic version of the PhIAT reagents demonstrated that a 10% difference in phosphorylation state could be determined between phosphopeptides in comparative samples.
2002. "Creating nanocavities of tunable sizes: Hollow helices." Proceedings of the National Academy of Sciences of the United States of America 99(18):11583-88. Abstract .
2002. "Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation." Electrophoresis 23:3143-3148. Abstract .
2002. "Genomes to Life "Center for Molecular and Cellular Systems": A research program for identification and characterization of protein complexes." OMICS. A Journal of Integrative Biology 6(4):287-303. Abstract Goal 1 of Department of Energy's Genomes to Life (GTL) program seeks to identify and characterize the complete set of protein complexes within a cell. Goal 1 forms the foundation necessary to accomplish the other objectives of the GTL program, which focus on gene regulatory networks and molecular level characterization of interactions in microbial communities. Together this information would allow cells and their components to be understood in sufficient detail to predict, test, and understand the responses of a biological system to its environment. The Center for Molecular and Cellular Systems has been established to identify and characterize protein complexes using high through-put analytical technologies. A dynamic research program is being developed that supports the goals of the Center by focusing on the development of new capabilities for sample preparation and complex separations, molecular level identification of the protein complexes by mass spectrometry, characterization of the complexes in living cells by imaging techniques, and bioinformatics and computational tools for the collection and interpretation of data and formation of databases and tools to allow the data to be shared by the biological community.
2002. "Low-Energy Collision-Induced Dissociation Fragmentation Analysis of Cysteinyl-Modified Peptides." Analytical Chemistry 74:2284-2292. Abstract The development of methods to chemically modify and isolate cysteinyl-residue containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjuction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of D. radiodurans, the presence of these labeled-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys-residue, and to differentiate identical Cys-peptides labeled with either ICAT-D0 or ICAT-D8.
2002. "Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry." Journal of Proteome Research 1(4):351-360. Abstract Currently, most proteomic studies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents, chaotropes, or reducing reagents that often interfere with electrospray ionization (ESI). To increase the identification of integral membrane proteins by LC-ESI-MS/MS, a sample preparation method combining carbonate extraction and surfactant-free organics solvent-assisted solubilization and proteolysis was developed and used to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic based on their positive grand average of hydropathicity values that covers 15% of the theoretical hydrophobic proteome. Using the PSORT algorithm, 268 identified proteins were recognized as integral membrane proteins covering 21% and 43% of the predicted integral cytoplasmic and outer membrane proteins, respectively. Of the integral cytoplasmic membrane proteins containing four or more predicted transmembrane domains (TMDs), 65% were identified by detecting at least one peptide spanning a TMD using LC-MS/MS. The extensive identification of highly hydrophobic proteins containing multiple TMDs confirms the efficacy of the described sample preparation protocol to isolate and solubilize integral membrane proteins and validates the method for large-scale analysis of bacterial membrane subproteomes using LC-ESI-MS/MS.
2002. "High-Throughput Global Peptide Proteomic Analysis by Combining Stable Isotope Amino Acid Labeling and Data-Dependent Multiplexed-MS/MS." Analytical Chemistry 74(19):4994-5000. Abstract .
2002. "Higher-Resolution Data-Dependent Selective External Ion Accumulation for Capillary LC-FTICR." International Journal of Mass Spectrometry 218(3):265-279. Abstract Data-dependent selective external ion ejection with improved resolution is demonstrated with a 3.5 tesla FTICR instrument employing DREAMS (Dynamic Range Enhancement Applied to Mass Spectrometry) technology. To correct for the fringing rf-field aberrations each rod of the selection quadrupole has been segmented into three sections, so that ion excitation and ejection was performed by applying auxiliary rf-only waveforms in the region of the middle segments. Two different modes of external ion trapping and ejection were studied with the mixtures of model peptides and a tryptic digest of bovine serum albumin. A mass resolution of about 100 has been attained for rf-only dipolar ejection in a quadrupole operating at a Mathieu parameter q of ~ 0.45. LC-ESI-DREAMS-FTICR analysis of a 0.1 mg/mL solution of bovine serum albumin digest resulted in detection of 82 unique tryptic peptides with mass measurement errors lower than 5 ppm, providing 100 % sequence coverage of the protein.
2002. "Higher-Resolution Data-Dependent Selective External ION Accumulation for Capillary LC-FTICR." International Journal of Mass Spectrometry 218(3):265-279. Abstract Data-dependent selective external ion ejection with improved resolution is demonstrated with a 3.5 tesla FTICR instrument employing DREAMS (Dynamic Range Enhancement Applied to Mass Spectrometry) technology. To correct for the fringing rf-field aberrations each rod of the selection quadrupole has been segmented into three sections, so that ion excitation and ejection was performed by applying auxiliary rf-only waveforms in the region of the middle segments. Two different modes of external ion trapping and ejection were studied with the mixtures of model peptides and a tryptic digest of bovine serum albumin. A mass resolution of about 100 had been attained for rf-only dipolar ejection in a quadrupole operating at a Mathieu parameter q of ~0.45. LC-ESI-DREAMS-FTICR analysis of a 0.1 mg/mL solution of bovine serum albumin digest resulted in detection of 82 unique tryptic peptides with mass measurement errors lower than 5 ppm, providing 100 % sequence coverage of the protein.
2002. "Toward a Human Blood Serum Proteome: Analysis by Multidimensional Separation Coupled with Mass Spectrometry." Molecular & Cellular Proteomics. MCP 1(12):947-955. Abstract Blood serum is a complex bodily fluid that contains proteins ranging in concentration over at least nine orders of magnitude. Using a combination of powerful mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with sub-mL quantities of serum, and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by online reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong-cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in an increase in the number of proteins detected in an individual serum sample by 3 to 5 fold. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/mL range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases, as a subjective measure of quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.
2001. "Sensitivity Enhancement of Electrospray Ionization MS for Aqueous Solutions in the Corona-Assisted Cone-Jet Mode." International Journal of Mass Spectrometry and Ion Processes 162(1-3):69-76. Abstract There is no abstract currently available for this item
2001. "Physical/Chemical Separations in the Break-up of Highly Charged Droplets from Electrosprays." Journal of the American Society for Mass Spectrometry 12(3):343-347. Abstract Highly charged droplets, as formed by an electrospray process, are known to undergo asymmetric fission to form smaller droplets. We have observed a chemical and physical separation phenomenon that occurs in the droplet break-up process and is related to a compound's surface activity in solution. Two experimental approaches demonstrated that the smaller satellite droplets and the progeny droplets generated by the spray formation process and asymmetric fission process to be surfactant-enriched. These smaller droplets were also effectively separated from the larger primary and residual droplets because of their smaller inertia and high surface charge density, and a region attributed to the initially formed smaller satellite droplets was found to be strikingly confined in a narrow periphery region of the electrospray. The phenomenon may have utility for chemical separations and have significant implications for the sensitivity and selectivity of electrospray ionization-mass spectrometry.
2001. "Generation of Multiple Electrosprays Using Microfabricated Emitter Arrays for Improved Mass Spectrometric Sensitivity." Analytical Chemistry 73(8):1658-1663. Abstract Arrays of micro-electrospray emitters were fabricated on polycarbonate substrates using a laser etching technique. Stable multi-electrosprays were successfully generated in the liquid flow rate range relevant to mass spectrometric applications. Comparison of electrosprays generated from the micro-fabricated emitter array and conventional fused silica capillaries showed similar spray characteristics and reliability. Higher total electrospray ion currents were observed as the number of electrosprays increased at a given total liquid flow rate. Consistent with the theoretical prediction, the total spray current at a constant total liquid flow rate was shown experimentally to be approximately proportional to the square root of the number of electrosprays. It is further projected that when total flow rate is optimized the maximum achievable total current will be proportioned to the number of emitters. Evaluation of the multi-electrospray device using a triple quadrople mass spectrometer showed a factor of 2 to 3 sensitivity enhancement for the spray numbers ranging from two to nine compared to a conventional single electrospray ionization source under the same operating conditions.
2001. "Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry." Electrophoresis 22:1652-1668. Abstract N/A
2001