pFind Studio: a computational solution for mass spectrometry-based proteomics
2014
Nature2014. Epshtein, V et al.
NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT:UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes.
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Nature2014. Epshtein, V et al.
NYU, Sch Med, Dept Biochem & Mol Pharmacol, New York, NY 10016 USA.
ABSTRACT:UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes.
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Analytical Chemistry2014. Zhao, Q et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Liaoning, Peoples R China.
ABSTRACT:Due to their extremely hydrophobic nature, the analysis of integral membrane proteins (IMPs) is of great challenge. Although various additives have been applied to improve the solubility of IMPs, they still suffer from low solubilization efficiency, incompatibility with trypsin digestion, or interference with MS detection. Herein, the systematic study on the effect of ionic liquid structure on membrane protein solubilization and trypsin biocompatibility was performed, based on which 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) was selected for the sample preparation of IMPs. Compared with other commonly used additives, such as sodium dodecyl sulfate (SDS), Rapigest, and methanol, C12Im-Cl showed the best performance. In addition, with a strong cation exchange trap column, it could be easily removed after trypsin digestion, which not only was beneficial to avoid protein precipitation during digestion but also had no adverse effect on LC-MS-based separation and detection. Such a C12Im-Cl-assisted sample preparation method was further applied to the membrane proteome analysis of rat brain. Compared with the SDS-assisted method, 1.4 and 3.5 times improvement on the identified IMP and hydrophobic peptide number were achieved (251 vs 178, and 982 vs 279). All these results demonstrated that the C12Im-Cl-assisted sample preparation method is of great promise to promote the large-scale membrane proteome profiling.
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Journal of proteome research2014. Cao, Liwei et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT:N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo H-f), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo H-f deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.
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Molecular & Cellular Proteomics2014. Liu, FY et al.
Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Peoples R China.
ABSTRACT:The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.
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Molecular & Cellular Proteomics2014. Fu, Y et al.
55 Zhongguancun East Rd, Beijing 100190, Peoples R China.
ABSTRACT:In shotgun proteomics, high-throughput mass spectrometry experiments and the subsequent data analysis produce thousands to millions of hypothetical peptide identifications. The common way to estimate the false discovery rate (FDR) of peptide identifications is the target-decoy database search strategy, which is efficient and accurate for large datasets. However, the legitimacy of the target-decoy strategy for protein-modification-centric studies has rarely been rigorously validated. It is often the case that a global FDR is estimated for all peptide identifications including both modified and unmodified peptides, but that only a subgroup of identifications with a certain type of modification is focused on. As revealed recently, the subgroup FDR of modified peptide identifications can differ dramatically from the global FDR at the same score threshold, and thus the former, when it is of interest, should be separately estimated. However, rare modifications often result in a very small number of modified peptide identifications, which makes the direct separate FDR estimation inaccurate because of the inadequate sample size. This paper presents a method called the transferred FDR for accurately estimating the FDR of an arbitrary number of modified peptide identifications. Through flexible use of the empirical data from a target-decoy database search, a theoretical relationship between the subgroup FDR and the global FDR is made computable. Through this relationship, the subgroup FDR can be predicted from the global FDR, allowing one to avoid an inaccurate direct estimation from a limited amount of data. The effectiveness of the method is demonstrated with both simulated and real mass spectra.
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Journal of Proteome Research2014. Yuan, ZF et al.
Univ Penn, Perelman Sch Med, Dept Biochem & Biophys, 3400 Civ Ctr,Bldg 421, Philadelphia, PA 19104 USA.
ABSTRACT:Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.
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Analytical Chemistry2014. Cao, QC et al.
Beijing Inst Radiat Med, Beijing Proteome Res Ctr, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT:Core fucosylation (CF) is a special glycosylation pattern of proteins that has a strong relationship with cancer. The Food and Drug Administration (FDA) has approved the core fucosylated alpha-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value. The major method for core fucosylated glycoprotein/glycopeptide analysis is neutral loss-based MS3 scanning under collision-induced dissociation (CID) by ion trap mass spectrometry. However, due to the limited speed and low resolution of the MS3 scan mode, it is difficult to achieve high-throughput, with only dozens of core fucosylated proteins identified in a single run. In this work, we developed a novel strategy for the identification of CF glycopeptides at a large scale, integrating the stepped fragmentation function, one novel feature of quadrupole-orbitrap mass spectrometry, with "glycan diagnostic ion"-based spectrum optimization. By using stepped fragmentation, we were able to obtain both highly accurate glycan and peptide information of a simplified CF glycopeptide in one spectrum. Moreover, the spectrum could be recorded with the same high speed as the conventional MS2 scan. By using the "glycan diagnostic ion"-based spectrum refinement method, the efficiency of the CF glycopeptide discovery was significantly improved. We demonstrated the feasibility and reproducibility of our method by analyzing CF glycoproteomes of mouse liver tissue and HeLa cell samples spiked with standard CF glycoprotein. In total, 1364 and 856 CF glycopeptides belonging to 702 and 449 CF glycoproteins were identified, respectively, within a 78-min gradient analysis, which was approximately a 7-fold increase in the identification efficiency of CF glycopeptides compared to the currently used method. In this work, we took core fucosylated glycopeptides as a practical example to demonstrate the great potential of our novel method for use in glycoproteome analysis, and we also anticipate using the flexible novel method in other research fields.
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Molecular & Cellular Proteomics2014. Lu, AP et al.
Tongji Univ, Sch Life Sci & Technol, Inst Prot Res, 1239 Siping Rd, Shanghai 200092, Peoples R China.
ABSTRACT:Conotoxins are peptide neurotoxins produced by predatory cone snails. They are mostly cysteine-rich short peptides with remarkable structural diversity. The conserved signal peptide sequences of their mRNA-encoded precursors have enabled the grouping of known conotoxins into a limited number of superfamilies. However, the conotoxins within each superfamily often present variable sequences, cysteine frameworks, and post-translational modifications. To understand better how conotoxins are diversified, we performed a venomic study with C. flavidus, an uninvestigated vermivorous Conus species, by combining transcriptomic and proteomic analyses. In order to obtain the full-length conotoxin sequences, protease digestion was not performed with the venom extraction prior to spectra acquisition via tandem mass spectrometry (MS/MS). Because conotoxins are produced from mRNA-encoded precursors by means of proteolytic cleavage, nonspecific digestion of precursors was applied during the database search. Special attention was also paid in interpreting the MS/MS spectra. All together, these analyses identified 69 nonredundant cDNA sequences and 31 conotoxin components with confident MS/MS spectra. A new Q-superfamily was also identified. More importantly, this study revealed that conotoxin-encoding transcripts are diversified by hypermutation, fragment insertion/deletion, and mutation-induced premature termination, and that a single mRNA species can produce multiple toxin products through alternative post-translational modifications and alternative cleavages of the translated precursor. These multiple diversification strategies at different levels may explain, at least in part, the diversity of conotoxins, and provide the basis for further investigation.
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Journal of Proteome Research2014. Chen, Z et al.
Chinese Acad Sci, Wuhan Inst Virol, State Key Lab Virol, Wuhan 430071, Peoples R China.
ABSTRACT:Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification used in signal transduction to control cell growth, proliferation, and stress responses. However, little is known about its extent and function in diatoms. Phaeodactylum tricornutum is a unicellular marine diatom that has been used as a model organism for research on diatom molecular biology. Although more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues have been predicted in P. tricornutum, no phosphorylation event has so far been revealed by classical biochemical approaches. Here, we performed a global phosphoproteomic analysis combining protein/peptide fractionation, TiO2 enrichment, and LC-MS/MS analyses. In total, we identified 264 unique phosphopeptides, including 434 in vivo phosphorylated sites on 245 phosphoproteins. The phosphorylated proteins were implicated in the regulation of diverse biological processes, including signaling, metabolic pathways, and stress responses. Six identified phosphoproteins were further validated by Western blotting using phospho-specific antibodies. The functions of these proteins are discussed in the context of signal transduction networks in P. tricornutum. Our results advance the current understanding of diatom biology and will be useful for elucidating the phosphor-relay signaling networks in this model diatom.
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