2009. "Evaluation of a High Intensity Focused Ultrasound-Immobilized Trypsin Digestion and 18 O-Labeling Method for Quantitative Proteomics." Analytical Chemistry 81(15):6272-6277. Abstract A new method that uses immobilized trypsin concomitant with ultrasonic irradiation results in ultra-rapid digestion and thorough 18O labeling for quantitative protein comparisons. The reproducible and highly efficient method provided effective digestions in <1 min and minimized the amount of enzyme required compared to traditional methods. This method was demonstrated for digestion of both simple and complex protein mixtures, including bovine serum albumin, a global proteome extract from bacteria Shewanella oneidensis, and mouse plasma, as well as for the labeling of complex protein mixtures, which validated the application of this method for differential proteomic measurements. This approach is simple, reproducible, cost effective, and rapid, and thus well-suited for automation.
2008. "High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex." Journal of Biological Chemistry 283(41):27829-27837. doi:10.1074/jbc.M803918200 Abstract Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of the photosynthetic electron transport chain in plants, algae, and cyanobacteria. Utilizing a high-throughput proteomic analysis of isolated PSII complexes from the cyanobacterium Synechocystis sp. PCC 6803, we have identified four PSII associated proteins that are encoded by the cofactor integration operon (cio). This operon contains genes with putative binding domains for chlorophyll, iron-sulfur centers, and bilins. Protein levels of this operon are more abundant in several PSII lumenal mutants, suggesting an accumulation of cio products in partially assembled PSII complexes. This provides a rare example of a bacterial operon whose protein products are translationally coordinated and associated with a single protein complex. Genetic deletion of cio results in decreased oxygen evolution by PSII, suggesting that cio products may function as regulators of PSII complex assembly or degradation, maybe facilitating an uncharacterized step in PSII assembly.
2008. "Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags." Analytical Chemistry 80(6):1871-82. doi:10.1021/ac702328x Abstract Identifying proteins correctly and with known levels of confidence remain as significant challenges for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR, and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and de novo-style data analyses of high precision MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3,132 confident peptide identifications (~5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3,359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed ~5% of the 3,359 identifications to be incorrect, and many more as potentially ambiguous, (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including non-tryptic peptides, peptides with various expected/unexpected chemical modifications, known/unknown posttranslational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.
2008. "Mass Spectrometry Analysis of Proteome-wide Proteolytic Post-translational Degradation of Proteins." Analytical Chemistry 80(15):5819-5828. doi:10.1021/ac800077w Abstract Protein proteolysis is an essential component to proper cell function. Here, we demonstrate a method for studying protein degradation by detection of intermediate intracellular peptides with a high-precision tandem mass spectrometry de novo sequencing-based approach. From a Saccharomyces cerevisiae lysate, we identified >1,200 peptides containing 6-100 amino acids without random false positives and ascribed most identifications as being products of protein degradation. Most protein degradation observed was located in the cytoplasm, and multiple types of cleavage were found to exist in addition to the expected trypsin-like and chymotrypsin-like preferences. The yeast nucleus was found as a proteolysis-inert organelle under the conditions studied and the V-ATPase to be degraded during disassembly. Additionally, matrix associated mitochondrial proteins functioning as transport carriers and gates were found to be commonly degraded. Determining these protein degradation events could eventually aid in understanding of cell biology and detection and treatment of protein degradation-related diseases.
2008. "De novo sequencing of unique sequence tags for discovery of post-translational modifications of proteins ." Analytical Chemistry 80(20):7742-7754. doi:10.1021/ac801123p Abstract De novo sequencing has a promise to discover the protein post-translation modifications; however, such approach is still in their infancy and not widely applied for proteomics practices due to its limited reliability. In this work, we describe a de novo sequencing approach for discovery of protein modifications through identification of the UStags (Anal. Chem. 2008, 80, 1871-1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry for peptides and polypeptides in a yeast lysate, and the de novo sequences obtained were filtered to define a more limited set of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags’ prefix and suffix sequences and the UStags themselves) were used to infer the possible sequence modifications. With this de novo-UStag approach, we uncovered some unexpected variances of yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. Random matching of the de novo sequences to the predicted sequences were examined with use of two random (false) databases, and ~3% false discovery rates were estimated for the de novo-UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity are described. The de novo-UStag complements the UStag method previously reported by enabling discovery of new protein modifications.
2008. "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. "Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor." Biochimica et Biophysica Acta--Proteins and Proteomics 1784(12):1935-1941. doi:10.1016/j.bbapap.2008.06.011 Abstract e(III) oxides are the most abundant source of reducible Fe(III) by microorganisms in most soils and sediments, yet few studies on the physiology of Fe(III)-reducing microorganisms during growth on Fe(III) oxide have been conducted because of the technical difficulties in working with cell growth and harvest in the presence of Fe(III) oxides. Geobacter sulfurreducens is a representative of the Geobacter species that predominate in a variety of subsurface environments in which Fe(III) oxide is important. In order to better understand the physiology of Geobacter species during growth on Fe(III) oxide, the proteome of G. sulfurreducens grown on Fe(III) oxide was compared with the proteome of cells grown with soluble Fe(III) citrate. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) revealed 19 proteins that were more abundant during growth on Fe(III) oxide than on soluble Fe(III). These included proteins related to protein synthesis, electron transfer and energy production, oxidative stress, protein folding, outer membrane proteins, nitrogen metabolism and hypothetical proteins. Further analysis of the proteome with the accurate mass and time (AMT) tag method revealed additional proteins associated with growth on Fe(III) oxide. These included the outer-membrane c-type cytochrome, OmcS and OmcG, which genetic studies have suggested are required for Fe(III) oxide reduction. Furthermore, several other cytochromes, as yet unstudied, were detected to be significantly up regulated during growth on Fe(III) oxide and other proteins of unknown function were more abundant during growth on Fe(III) oxide than on soluble Fe(III). PilA, the structural protein for pili, which is required for Fe(III) oxide reduction, and other pilin-associated proteins were also more abundant during growth on Fe(III) oxide. Confirmation of the differential expression of proteins known to be important in Fe(III) oxide reduction was observed, and an additional number of previously unidentified proteins were found with significant abundance in the cells grown under conditions of Fe(III) oxide reduction.
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.
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. "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. "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. "The Proteome of Dissimilatory Metal-reducing Microorganism Geobacter Sulfurreducens under Various Growth Conditions." Biochimica et Biophysica Acta--Proteins and Proteomics 1764(7):1198-1206. doi:10.1016/j.bbapap.2006.04.017 Abstract The global protein analysis of Geobacter sulfurreducens, a model for the Geobacter species that predominate in many Fe(III)-reducing subsurface environments, was characterized with ultra high pressure liquid chromatography and mass spectrometry using accurate mass and time (AMT) tags as well as with more traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Cells were grown under eight different growth conditions in order to enhance the potential that genes would be expressed. Over 3,187 gene products, representing about 92% of the total predicted gene products in the genome, were detected. The AMT approach was able to identify a much higher number of proteins than could be detected with the 2-D PAGE approach. A high proportion of predicted proteins in most protein role categories were detected with the highest number of proteins identified in the hypothetical protein role category. Furthermore, 91 c-type cytochromes of 111 predicted genes in the G. sulfurreducens genome were identified. Localization studies indicated that computational predictions of cytochrome location were limited. Differences in the abundance of cytochromes and other proteins under different growth conditions provided information for future functional analysis of these proteins. These results demonstrate that a high percentage of the predicted proteins in the G. sulfurreducens genome are produced and that the AMT approach provides a rapid method for comparing differential expression of proteins under different growth conditions in this organism.
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. "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.
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. "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.
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. "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 .
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. "Evaluation of Enzymatic Digestion and Liquid Chromatography-Mass Spectrometry Peptide Mapping of the Integral Membrane Protein Bacteriorhodopsin." Electrophoresis 23(18):3224-3232. Abstract .
1999. "High Throughput Proteome-Wide Precision Measurements of Protein Expression using Mass Spectrometry." Journal of the American Chemical Society 121(34):7949-7950.