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. "Identification of a putative protein profile associating with tamoxifen therapy resistance in breast cancer." Molecular & Cellular Proteomics. MCP 8(6):1278-1294. doi:10.1074/mcp.M800493-MCP200 Abstract Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.
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. "Platelet Proteome Changes Associated with Diabetes and During Platelet Storage for Transfusion." Journal of Proteome Research 8(5):2261-2272. Abstract Human platelets play a key role in homeostasis and thrombosis and have recently emerged as key regulators of inflammation. Platelets stored for transfusion produce pro-thrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, a mass spectrometry based proteomics study was performed on platelets collected from healthy and type-2 diabetics stored for transfusion. Strikingly, our innovative and sensitive proteomic approach identified 146 proteins that were either up- or down-regulated in type-2 diabetics relative to non-diabetic controls, 151 proteins whose abundances changed during a 5-day storage period and 22 proteins whose abundance changed after 5-days of storage were only observed in samples from diabetics. Notably our studies are the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. These identified differences allow us to formulate new hypotheses and experimentation to improve clinical outcomes by targeting "high risk platelets" that render platelet transfusion less effective or even unsafe.
2009. "Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II ." Molecular Cell 33(2):192-203. doi:doi:10.1016/j.molcel.2008.12.015 Abstract In addition to RNA polymerases I, II and III, which are multi-subunit RNA polymerases found in all eukaryotes, plants have catalytic subunits for two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V (formerly Pol IVa and Pol IVb, respectively). Pol IV and Pol V play non-redundant roles in siRNA-directed DNA methylation and gene silencing pathways.
2008. "High-throughput proteomics of breast carcinoma cells: a focus on FTICR-MS." Expert Review of Proteomics 2008 Jun;5(3):445-55. Abstract Discovery of better biomarkers for diagnosis, prognosis, and therapy-response prediction is the most critical task of a scientific quest aimed at developing newly designed, tailor-made therapies for patients with cancer. Consequently, a proteome wide analysis, in addition to genomic studies, is an absolute requirement for a complete functional understanding of tumor biology. Ultra-sensitive, high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) currently holds an important role in fulfilling the demands of biomarker discovery. In this review, we describe the applicability of FTICR MS for breast cancer proteomics, particularly for the analysis of complex protein mixtures obtained from a limited number of cells typically available from clinical specimens.
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. "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. "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.
2007. "NanoLC-FT-ICR MS improves proteome coverage attainable for ~3000 laser microdissected breast carcinoma cells." Proteomics 7(2):323-329. doi:10.1002/pmic.200600293 Abstract Genomics and proteomics assays hold great promise for unrevealing molecular events that underlie human disease. Essential to this quest is the ability to effectively analyze clinical samples, but this task is considerably complicated by tissue heterogeneity. Laser capture microdissection (LCM) can be used to selectively isolate targeted cell populations (such as tumor cells) from their native tissue environment. However, the small number of cells that are typically procured by LCM severely limits the proteome coverage and biomarker discovery potential achievable by conventional proteomics platforms. Herein, we report on the use of a nano liquid chromatography-Fourier transform ion clyclotron resonance mass spectrometry (nLC-FTICR MS) platform for analyzing protein digests of approximately 3,000 LCM-derived tumor cells from breast carcinoma tissue, which corresponds to approximately 300 ng of total protein. A total of 2,836 peptides were identified by matching LC-MS data to accurate mass and time (AMT) tag databases that were previously established for the human mammary epithelium and several breast cancer cell lines. The peptide identifications correspond to 1,139 unique proteins confidently identified with 2 or more peptides. Based on categorization by Gene Ontology, identified proteins appear to cover a wide variety of biological functions and cellular compartments. This work demonstrates that a substantial number of proteins can be identified from a limited number of cells using the AMT tag approach and opens a door for high throughput in-depth proteomics analysis of clinical samples.
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. "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. "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.
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. "Structural Studies of Vγ2Vδ2 T Cell Phosphoantigens." Chemistry & Biology 13(9):985-992. doi:10.1016/j.chembiol.2006.08.007 Abstract Human γδ T cells containing the Vγ2Vδ2 (Vγ9Vδ2) T cell receptor are stimulated by a broad variety of small, phosphorus-containing antigenic molecules called “phosphoantigens”. The structures of several species present in both Mycobacteria (TUBags1-4) and in E. coli and have been reported to contain a formyl-alkyl diphosphate core. Here, we report the synthesis of the lead member of the series, 3-formyl-1-butyl diphosphate. This compound has low activity for γδ T cell stimulation, unlike its highly active isomer (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), necessitating a revision of the structure of TUBag1. Likewise, the structure of the species identified as the pentyl analog (TUBag 2) is revised to 6-phosphogluconate. These results indicate that neither TUBag1 nor the m/e 275 species proposed for TUBag2 are 3-formyl-1-alkyl diphosphates, leading to the conclusion that none of the natural phosphoantigens (TUBags1-4) possess the structures reported previously.
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.
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. "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. "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.
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. "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. "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. "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. "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. "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.
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. "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. "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. "Synthesis and Characterisation of Pd(II) Complexes with a Derivative of Aminoazobenzene - Dynamic H-1-NMR Study of Cyclopalladation Reactions in DMF." Journal of Organometallic Chemistry 687(1):85-99. Abstract Three new Pd(II) complexes, i.e. [PdCl2L]2 (A), PdCl2L2 (B) and [Pd(μ-Cl)(L-H)]2 (C), each with two diethyl [α-(4-benzenazoanilino)-2-hydroxybenzyl]phosphonates (L) bound to either one or two palladium atoms, are synthesized and characterized by elemental analysis, by IR, UV–vis and solid-state 13C-NMR spectra. Complexes B and C are additionally characterized by 1H-, 13C- and 31P-NMR and electrospray mass spectrometry (ESMS) studies using dimethylformamide (DMF) as a solvent. In DMF solution adducts A and B undergo spontaneous rearrangement into the cyclopalladated complex C. Dynamic 1H-NMR study of this rearrangement as well as of the reactions of L with PdCl2 and Na2PdCl4 revealed a complex equilibrium in DMF solutions and enabled the formation mechanism of all involved species to be resolved. The complex A is immediately solvolyzed producing two molecules of intermediate M [PdCl2(L)(DMF)]. Complex M was also the first intermediate in the reaction of L with PdCl2. Once present in concentration above 10−5 mol dm−3 M dimerizes very fast into chloro-bridged dimer [PdCl(μ-Cl)(L)]2 (D) which undergoes cyclopalladation and converts into the complex C. The formation of C from the intermediate D is clearly demonstrated by the concentration dependence of the cyclopalladation reaction which has order greater than one. Chloride ions, released by cyclopalladation, react with D by splitting chloro-bridge and binding to metal atoms to produce byproduct [PdCl3(L)]− (T). The same species T are formed in the reaction of L with Na2PdCl4 whereby a chloride ion is replaced by the ligand L. The complex B undergoes similar, but slower, solvolytic reaction producing M and L while further reaction steps are identical as in the solvolysis of A.
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. "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. "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. "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. "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. "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. "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 .
2001. "Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry." Electrophoresis 22:1652-1668. Abstract N/A
2001. "Packed Capillary Reversed-Phase Liquid Chromatography with High Performance Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Proteomics ." Analytical Chemistry 73(8):1766-1775. Abstract In this study, high efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-um) C18 bonded particles provided a total peak capacity of ~1,000 for cellular proteolytic polypeptides when connecting to FTICR mass spectrometer through electrospray interface under composition gradient conditions at a pressure of 10,000 psi. Two-dimensional analyses from combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of >1 million polypeptide components, and simultaneously provided information for the identification of the seperated compents based upon the accurate mass tag concept previously described. For Deinococcus radiodurans tryptic digests, ~48.6000 polypeptides from ~60,7000 isotopic distributions have been detected in a single run. Large amount (e.g., 500 ug) of cellular proteolytic digests could be loaded on packed capillaries (e.g., 150-um inner diameter) without significant loss of speration efficiency. Pre-columns with suitable inner diameters were found useful for improving the elution reproducibility and without significant loss of seperation quality. Porous particle packled capillaries are more favorable than those containing non-porous particles because of their high sample capacity even for seperations of cellular proteolutic polypeptides with a moderate amount sample loading (e.g., 50 ug protein content).
2001. "High-Throughput Proteomics Using High Efficiency Multiple-Capillary Liquid Chromatography With On-Line High-Performance ESI FTICR Mass Spectrometry." Analytical Chemistry 73(13):3011-3021. Abstract We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system was operated at the pressure of 10,000 psi using commercial LC pumps to deliver the mobile phase and newly developed passive feedback valves to switch the mobile phase flow and introduce samples. The multiple-capillary LC system was composed of several serially connected dual-capillary column devices. The dual-capillary column approach was designed to eliminate the time delay for regeneration (or equilibrium) of the capillary column after its use under the mobile phase gradient condition (i.e. one capillary column was used in separation and the other was washed using mobile phase A). The serially connected dual-capillary columns and ESI sources were operated independently, and could be used for either "backup" operation or with other mass spectrometer(s). This high-efficiency multiple-capillary LC system uses switching valves for all operations and is highly amenable to automation. The separations efficiency of dual-capillary column device, optimal capillary dimensions (column length and packed particle size), suitable mobile phases for electrospray, and the capillary re-generation were investigated. A high magnetic field (11.5 tesla) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system through an electrospray ionization source. The capillary LC provided a peak capacity of ~600, and the 2-D capillary LC-FTICR provided a combined resolving power of >6 x 10 7 polypeptide isotopic distributions. For yeast cellular tryptic digests, >100,000 polypeptides were typically detected, and ~1,000 proteins can be characterized in a single run.
2001. "High-Throughput Peptide Identification from Protein Digests Using Data-Dependent Multiplexed Tandem FTICR Mass Spectrometry Coupled with Capillary Liquid Chromatography." Analytical Chemistry 73(14):3312-3322. Abstract Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociatioin pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysates). Using "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several co-eluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high throughput analysis of a complex bacterial proteome.
2001. "Basicity of the Amino Groups of the Aminoglycoside Amikacin Using Capillary Electrophoresis and coupled CE-MS-MS techniques." Analytical Chemistry 73(16):4028-4036. Abstract This paper describes the use of capillary electrophoresis (CE) and coupled CE and mass spectrometric techniques (CE-MS-MS) to measure the values of the pKa of the amino groups of small molecules -- of the aminoglycoside antibiotic amikacin, and of its acetylated derivatives. These values of pKa (8.4, 6.7, 9.7, and 8.4) were determined by measuring the electrophoretic mobilities of the molecules as a function of pH; they are within 0.7 units of certain values reported in the literature (by 13C and 15N NMR spectroscopies [Gaggelli, 1995 #805] [Cox, 1997 #806]), and resolved ambiguities left by these earlier studies. The observed variation in the values of pKa of an amino group in different acetylated derivatives also indicated the complex dependence of the degree of ionization on the environment of the ionizable group.
2001. "Quantitative Analysis of Bacterial and Mammalian Proteomes using a Combination of Cysteine Affinity Tags and 15N-Metabolic Labeling." Analytical Chemistry 73(9):2132-2139. Abstract N/A
2000. "Stepwise Mobilization of Focused Proteins in Capillary Isoelectric Focusing Mass Spectrometry." Analytical Chemistry 72(7):1462-1468.
2000. "Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Analysis of Large Polymerases Chain Reaction Products." Journal of the American Society for Mass Spectrometry 11(4):333-337. Abstract We have attempted to expand the size range of PCR products that can be analyzedby electroscopy ionization (ESI) Fourier transformion cyclotron resonance (FTICR) mass spectrometry. The mass measurement accruacy obtained illustrates that a signel base substitution could be identified at the size of PCR product with a 7 tesla ESI-FTICR
2000. "Proteome Analysis using Selective Incorporation of Isotopically Labeled Amino Acids." Journal of the American Society for Mass Spectrometry 11(1):78-82. Abstract A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins were extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), 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 Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of using this method to unambiguously identify proteins isolated from E. coli.
2000. "Accurate Mass Multiplexed Tandem Mass Spectrometry for High-Throughput Polypeptide Identification from Mixtures." Analytical Chemistry 72(8):1918-1924. Abstract We report a new tandem mass spectrometric approach for the improved identification of polypeptides from mixtures (e.g. using genomic databases). The approach involves the dissociation of several species simultaneously in a single experiment and provides both increased speed and sensitivity. The data analysis makes use of the known fragmentation pathways for polypeptides and highly accurate mass-measurements for both the set of parent polypeptides and their fragments. The accurate mass information makes it possible to attribute most fragments to a specific parent species. We provide an initial demonstration of this multiplexed tandem MS approach using an FTICR mass spectrometer with a mixture of seven polypeptides dissociated using infrared irradiation from a CO2 laser. The peptides are successfully identified from the largest genomic data base yet available (C. elegans) which is equivalent in complexity to that for a specific type of differentiated cell in the human genome. Additionally, since only a few enzymatic fragments are necessary to identify unambiguously a protein from an appropriate database, it is anticipated that the multiplexed MSIMS method will allow the more rapid identification of proteins after online separation of mixtures of their enzymatically produced polypeptides.
2000. "Separation and Detection of Intact Noncovalent Protein Complexes from Mixtures by On-Line Capillary Isoelectric Focusing-Mass Spectrometry." Analytical Chemistry 72(21):5356-5360. Abstract Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During hte mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for anlysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.
2000. "Characterization of Human Alcohol Dehydrogenase Isoenzymes by Capillary Isoelectric Focusing-Mass Spectrometry." Electrophoresis 21(12):2368-2375. Abstract The human liver alcohol dehydrogenase isoenzymes (ADH) are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (PI) values(the 13 possible isoenzymes having pIs in the range of 8.26- 8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance(ESI FTICR) mass spectrometry was applied to separate and characterize mixtures of aa, B1B1, and B3B3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range, and having pI differences as small as ~0.02 pH units. ESI-FTICR analysis of native ADHs revealed that each non-covalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.
2000. "Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometric Analysis of the Recombinant Human Macrophage Colony Stimulating Factor Beta and Derivatives." Journal of the American Society for Mass Spectrometry 11(3):237-243. Abstract The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor B (rhM-CSF B) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (~25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.
2000. "Mass Spectrometric Detection for Capillary Isoelectric Focusing Separations of Complex Protein Mixtures." Electrophoresis 21(7):1372-1380. Abstract Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of ~300 ng protein in a single CIEF-FTICR analysis of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.
2000. "Protein Identification with a Single Accurate Mass of a Cysteine-Containing Peptide and Constrained Database Searching." Analytical Chemistry 72(6):1112-1118. Abstract A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.
2000. "Two Dimensional Electrophoretic/Chromatographic Separations Combined with Electrospray Ionization FTICR Mass Spectrometry for High Throughput Proteome Analysis." Journal of Microcolumn Separations 12(7):383-390. Abstract N/A
2000. "Utility of Accurate Mass Tags for Proteome-Wide Protein Identification." Analytical Chemistry 72(14):3349-3354. Abstract An enabling capability for proteomics would be the ability to study protein expression on a global scale. While several different separation and analysis options are being investigated to aid proteomics, mass spectrometry (MS) is rapidly becoming the core instrumental technology used to characterize the large number of proteins that constitute a proteome. To be most effective, proteomic measurements must have high-throughput capabilities, ideally allowing thousands of proteins to be identified on a time-scale of hours. Most strategies of identification by MS rely on the analysis of enzymatically produced peptides originating from an isolated protein followed by either by peptide mapping or tandem MS (MS/MS) to obtain sequence information for a single peptide. In the case of peptide mapping, several peptide masses are needed to unambiguously identify a protein with the typically achieved mass measurement accuracies (MMA). The ability to identify proteins based on the masse of a single peptide (i.e. an accurate mass tag; AMT) is proposed, and is largely dependent on the MMA that can be achieved. To determine the MMA necessary to enable the use of AMTs for proteome-wide protein identification, we analyzed the predicted proteins and their tryptic fragments from Saccharomyces cerevisiae and Caenorhabditis elegans. The results show that low ppm (i.e., -1 ppm) level measurements have practical utility for analysis of small proteomes. Additionally, up to 85% of the peptides predicted from these organisms can function as AMTs at sub-ppm MMA levels attainable using Fourier transform ion cyclotron resonance MS. Additional information, such as sequence constraints, should enable even more complex proteomes to be studied at more modest mass measurement accuracies. Once AMTs are established, subsequent high throughput measurements of proteomes should be greatly facilitated.
2000. "Obtaining More Accurate Fourier Transform Ion Cyclotron Resonance Mass Measurements Without Internal Standards Using Multiply Charged Ions." Journal of the American Society for Mass Spectrometry 11(5):416-421. Abstract Space charge effects produce frequency shifts in Fourier transfoffi1 ion cyclotron resonance (FTICR) mass spectrometry and correction for these shifts is necessary for obtaining accurate mass measurements. We report a novel method for ~btaining accurate mass calibration to correct for space-charge induced mass shifts without the requirement for internal calibrants. The new approach is particularly well suited for electrospray ionization-FTICR mass spectra that contain multiple charge states of the same molecular species. This method, Deconvolution of Coulombic Affected Linearity (DeCAL ), is described and presented with several examples demonstrating the increased mass measurement accuracy obtained. DeCAL will allow more routine high mass accuracy measurements to be made in conjunction with chromatographic separations for complex mixture analysis, obviating the need for internal calibration in many applications.
1999. "Electrospray Ionization-Mass Spectrometry Study of the Interaction of Cisplatin-Adducted Oligonucleotides with Human XPA Minimal Binding Domain Protein." Analytical Biochemistry 272(1):26-33. Abstract We have used electrospray ionization (ESI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to examine the cisplatin-adducted oligonucleotide and its interation with the XPA minimal binding doman (XPA-MBD).
1999. "High Throughput Proteome-Wide Precision Measurements of Protein Expression using Mass Spectrometry." Journal of the American Chemical Society 121(34):7949-7950.
1999. "Egg-Laying Hormone Peptides in the Aplysiidae Family." Journal of Experimental Biology 202:2961-2973. Abstract The neuropeptidergic bag cells of the marine mollusc "Aplysia californica" are involved in the egg-laying behavior of the animal. These neurosecretory cells synthesize and egg-laying hormone (ELH) precursor protein, yielding multiple bioactive pertides, including ELH, several bag cell peptides (BCP) and acidic peptide (AP). While immunohistochemical studies have involved a number of species, homologous peptides have been biochemically characterized in relatively few Aplysiidae species. In this study, a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and electrospray ionization Fourier transform ion cyclotron resonance MS is used to characterize and compare the ELH peptides from related opithobranch molluscs including "Aplysia vaccaria and Phyllaplysia taylori". The peptide profiles of bag cells from these two Aplysiidae species are similar to that of "A. californica" bag cells. In an effort to characterize further several of these peptides, peptides from multiple groups of cells of each species were extracted, and microbore liquid chromatography was used to separate and isolate them. Several MS-based sequencing approaches are applied to obtain the primary structures of bag cell peptides and ELH. Our studies reveal that a-BCPs are 100% conserved across all species studied. In addition, the complete sequences of e-BCP and ELH of A. vaccaria were determined. They show a high degree of homology to their counterparts in a. californica, with only a few amino acid residue substitutions.
1999. "Probing Proteomes Using Capillary Isoelectric Focusing-Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." Analytical Chemistry 71(11):2076-2084. Abstract We describe progress in the development and initial application of the poweful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the preteome of the model system Escherichia coli.
1999. "Pressure Limited Sustained Off-Resonance Irradiation for Collision-Activated Dissociation in Fourier Transform Mass Spectrometry." Journal of the American Society for Mass Spectrometry 10:15-18. Abstract A theoretical evalution of the sustained off-resonance irradiation of ions (SORI) in the presence of a collisional buffer gas in a Fourier transform ion cyclotron resonance mass spectrometer is presented.
1999. "Retrieval of DNA using Soft-Landing after Mass Analysis by ESI-FTICR for Enzymatic Manipulation." Journal of the American Chemical Society 121(38):8961-8962. Abstract A novel strategy has been developed for the analysis, separation and retrieval of oligonucleotides based upon the soft-landing of ions and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Stored waveform inverse Fourier transform was used to isolate selected DNA segments from a mixture prior to collection on a membrane surface. The soft-landing DNA was amplified by polymerase chain reaction (PCR) and analyzed by conventional gel electrophoresis. FTICR was also used to confirm the identity of the PCR products. This methodology allows the study of modified or damaged DNA as well as sequence variants present al low relative abundances, and is of particular value where characterization is impractical using conventional protocols.