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. "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. "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.
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. "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. "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. "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. "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. "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. "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.
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. "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. "Protein Composition of the Vaccinia Virus Mature Virion." Virology 358(1):233-247. Abstract The protein content of vaccinia virus mature virions, purified by rate zonal and isopycnic centrifugation and solubilized by SDS or a solution of urea and thiourea, was determined by the accurate mass and time tag technology which uses both tandem mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry to detect tryptic peptides separated by high-resolution liquid chromatography. Eighty vaccinia virus-encoded proteins representing 37% of the 218 genes annotated in the complete genome sequence were detected in at least three analyses. Ten proteins accounted for approximately 80% of the mass, while the least abundant proteins made up 1% or less of the mass. Thirteen identified proteins were not previously reported as components of virions. On the other hand, 8 previously described virion proteins were not detected here, presumably due to technical reasons including small size and hydrophobicity. In addition to vaccinia virus-encoded proteins, 24 host proteins omitting isoforms were detected. The most abundant of these were cytoskeletal proteins, heat shock proteins, and proteins involved in translation.
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.
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. "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. "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. "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. "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. "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.
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. "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. "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. "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. "Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks." Journal of Proteome Research 4(2):268-274. doi:10.1021/pr049847a Abstract Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.
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.
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. "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. "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. "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. "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. "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.
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. "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. "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.