Mapping the Subcellular Proteome of <i>Shewanella oneidensis</i> MR-1 using Sarkosyl-Based Fractionation and LC−MS/MS Protein Identification

A simple and effective subcellular proteomic method for fractionation based on osmotic lysis, differential centrifugation, and Sarkosyl solubilization was applied to the Gram-negative bacterium Shewanella oneidensis to gain insight into its subcellular architecture. Global differences in bacterial cytoplasm, inner membrane, periplasm, and outer membrane protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution liquid chromatography−tandem mass spectrometry. Proteins predicted to be localized to each subcellular fraction were enriched ∼2-fold (on average) in each fraction compared to crude cell lysates. In addition, the Sarkosyl solubilization method facilitated separation of the inner and outer membranes, making the procedure amenable for effective probing of the subcellular proteome of Gram-negative bacteria via liquid chromatography−tandem mass spectrometry. With 40% of the observable proteome represented, this study provides extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein−membrane interactions in other Gram-negative bacteria

Global Whole-Cell FTICR Mass Spectrometric Proteomics Analysis of the Heat Shock Response in the Radioresistant Bacterium <i>Deinococcus </i><i>r</i><i>adiodurans</i>

The results of previous studies indicated that D. radiodurans mounts a regulated protective response to heat shock, and that expression of more than 130 genes, including classical chaperones such as the groESL and dnaKJ operons and proteases such as clpB are induced in response to elevated temperature. In addition, previous qualitative whole-cell mass spectrometric studies conducted under heat shock conditions indicated global changes in the D. radiodurans proteome. To enable the discovery of novel heat shock inducible proteins as well as gain greater biological insight into the classical heat shock response at the protein level, we undertook the global whole-cell FTICR mass spectrometric proteomics study reported here. We have greatly increased the power of this approach by conducting a large number of replicate experiments in addition to taking a semiquantitative approach to data analysis, finding good reproducibility between replicates. Through this analysis, we have identified with high confidence a core set of classical heat shock proteins whose expression increases dramatically and reproducibly in response to elevated temperature. In addition, we have found that the heat shock proteome includes a large number of induced proteins that have not been identified previously as heat responsive, and have therefore been designated as candidate responders. Finally, our results are consistent with the hypothesis that elevated temperature stress could lead to cross-protection against other related stresses. Keywords: heat shock • Deinococcus radiodurans • FTICR proteomic

Proteome Analysis of <i>Desulfovibrio desulfuricans</i> G20 Mutants Using the Accurate Mass and Time (AMT) Tag Approach

Abundance values obtained from direct LC−MS analyses were used to compare the proteomes of six transposon-insertion mutants of Desulfovibrio desulfuricans G20, the lab strain (G20lab) and a sediment-adapted strain (G20sediment). Three mutations were in signal transduction histidine kinases, and three mutations were in other regulatory proteins. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to analyze the proteomes. A total of 1318 proteins was identified with high confidence, approximately 35% of all predicted proteins in the D. desulfuricans G20 genome. Proteins from all functional categories were identified. Significant differences in the abundance of 30 proteins were detected between the G20lab strain and the G20sediment strain. Abundances of proteins for energy metabolism, ribosomal synthesis, membrane biosynthesis, transport, and flagellar synthesis were affected in the mutants. Specific examples of proteins down-regulated in mutants include a putative tungstate transport system substrate-binding protein and several proteins related to energy production, for example, 2-oxoacid:acceptor oxidoreductase, cytochrome c-553, and formate acetyltransferase. In addition, several signal transduction mechanism proteins were regulated in one mutant, and the abundances of ferritin and hybrid cluster protein were reduced in another mutant. However, the similar abundance of universal stress proteins, heat shock proteins, and chemotaxis proteins in the mutants revealed that regulation of chemotactic behavior and stress regulation might not be observed under our growth conditions. This study provides the first proteomic overview of several sediment fitness mutants of G20, and evidence for the difference between lab strains and sediment-adapted strains at the protein level. Keywords: Desulfovibrio desulfuricans G20 • accurate mass and time (AMT) tag • proteome of mutants • regulatory gene

Proteomics-based Compositional Analysis of Complex Cellulase–Hemicellulase Mixtures

Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases, and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase-producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Here, we show that proteomics-based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements toward commercialization of plant biomass-derived fuels and chemicals

Large Improvements in MS/MS-Based Peptide Identification Rates using a Hybrid Analysis

We report a hybrid search method combining database and spectral library searches that allows for a straightforward approach to characterizing the error rates from the combined data. Using these methods, we demonstrate significantly increased sensitivity and specificity in matching peptides to tandem mass spectra. The hybrid search method increased the number of spectra that can be assigned to a peptide in a global proteomics study by 57−147% at an estimated false discovery rate of 5%, with clear room for even greater improvements. The approach combines the general utility of using consensus model spectra typical of database search methods with the accuracy of the intensity information contained in spectral libraries. A common scoring metric based on recent developments linking data analysis and statistical thermodynamics is used, which allows the use of a conservative estimate of error rates for the combined data. We applied this approach to proteomics analysis of Synechococcus sp. PCC 7002, a cyanobacterium that is a model organism for studies of photosynthetic carbon fixation and biofuels development. The increased specificity and sensitivity of this approach allowed us to identify many more peptides involved in the processes important for photoautotrophic growth

Proteome Analysis of <i>Desulfovibrio desulfuricans</i> G20 Mutants Using the Accurate Mass and Time (AMT) Tag Approach

Abundance values obtained from direct LC−MS analyses were used to compare the proteomes of six transposon-insertion mutants of Desulfovibrio desulfuricans G20, the lab strain (G20lab) and a sediment-adapted strain (G20sediment). Three mutations were in signal transduction histidine kinases, and three mutations were in other regulatory proteins. The high-throughput accurate mass and time (AMT) tag proteomic approach was utilized to analyze the proteomes. A total of 1318 proteins was identified with high confidence, approximately 35% of all predicted proteins in the D. desulfuricans G20 genome. Proteins from all functional categories were identified. Significant differences in the abundance of 30 proteins were detected between the G20lab strain and the G20sediment strain. Abundances of proteins for energy metabolism, ribosomal synthesis, membrane biosynthesis, transport, and flagellar synthesis were affected in the mutants. Specific examples of proteins down-regulated in mutants include a putative tungstate transport system substrate-binding protein and several proteins related to energy production, for example, 2-oxoacid:acceptor oxidoreductase, cytochrome c-553, and formate acetyltransferase. In addition, several signal transduction mechanism proteins were regulated in one mutant, and the abundances of ferritin and hybrid cluster protein were reduced in another mutant. However, the similar abundance of universal stress proteins, heat shock proteins, and chemotaxis proteins in the mutants revealed that regulation of chemotactic behavior and stress regulation might not be observed under our growth conditions. This study provides the first proteomic overview of several sediment fitness mutants of G20, and evidence for the difference between lab strains and sediment-adapted strains at the protein level. Keywords: Desulfovibrio desulfuricans G20 • accurate mass and time (AMT) tag • proteome of mutants • regulatory gene

Proteomics-based Compositional Analysis of Complex Cellulase–Hemicellulase Mixtures

Efficient deconstruction of cellulosic biomass to fermentable sugars for fuel and chemical production is accomplished by a complex mixture of cellulases, hemicellulases, and accessory enzymes (e.g., >50 extracellular proteins). Cellulolytic enzyme mixtures, produced industrially mostly using fungi like Trichoderma reesei, are poorly characterized in terms of their protein composition and its correlation to hydrolytic activity on cellulosic biomass. The secretomes of commercial glycosyl hydrolase-producing microbes was explored using a proteomics approach with high-throughput quantification using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Here, we show that proteomics-based spectral counting approach is a reasonably accurate and rapid analytical technique that can be used to determine protein composition of complex glycosyl hydrolase mixtures that also correlates with the specific activity of individual enzymes present within the mixture. For example, a strong linear correlation was seen between Avicelase activity and total cellobiohydrolase content. Reliable, quantitative and cheaper analytical methods that provide insight into the cellulosic biomass degrading fungal and bacterial secretomes would lead to further improvements toward commercialization of plant biomass-derived fuels and chemicals

Carbohydrate active enzymes (CAZymes) and fibro-slime proteins observed from proteomes extracted from extracellular medium, outer membrane, and periplasm fractions.

<p>EM = extracellular medium, PE = periplasm, UNIQUE = observed in only one fraction and in all of the three replicates for that fraction.</p><p>Carbohydrate active enzymes (CAZymes) and fibro-slime proteins observed from proteomes extracted from extracellular medium, outer membrane, and periplasm fractions.</p

A Data-Mining Scheme for Identifying Peptide Structural Motifs Responsible for Different MS/MS Fragmentation Intensity Patterns

Although tandem mass spectrometry (MS/MS) has become an integral part of proteomics, intensity patterns in MS/MS spectra are rarely weighted heavily in most widely used algorithms because they are not yet fully understood. Here a knowledge mining approach is demonstrated to discover fragmentation intensity patterns and elucidate the chemical factors behind such patterns. Fragmentation intensity information from 28 330 ion trap peptide MS/MS spectra of different charge states and sequences went through unsupervised clustering using a penalized K-means algorithm. Without any prior chemistry assumptions, four clusters with distinctive fragmentation patterns were obtained. A decision tree was generated to investigate peptide sequence motif and charge state status that caused these fragmentation patterns. This data-mining scheme is generally applicable for any large data sets. It bypasses the common prior knowledge constraints and reports on the overall peptide fragmentation behavior. It improves the understanding of gas-phase peptide dissociation and provides a foundation for new or improved protein identification algorithms

Statistical Characterization of the Charge State and Residue Dependence of Low-Energy CID Peptide Dissociation Patterns

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx−Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities