pFind Studio: a computational solution for mass spectrometry-based proteomics
2017
Frontiers in plant science2017. Zhang, Yunxiang et al.
Chinese Acad Sci, Inst Mt Hazards & Environm, Key Lab Mt Surface Proc & Ecol Regulat, Chengdu, Peoples R China
ABSTRACT:With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteome analysis was performed in P. cathayana females and males. A total of 2,405 proteins were identified, with 331 proteins defined as differentially expressed proteins (DEPs). Among of these, 79 and 138 DEPs were decreased and 47 and 107 DEPs were increased under high solar UV-B radiation in females and males, respectively. A bioinformatics analysis categorized the common responsive proteins in the sexes as related to carbohydrate and energy metabolism, translation/transcription/post-transcriptional modification, photosynthesis, and redox reactions. The responsive proteins that showed differences in sex were mainly those involved in amino acid metabolism, stress response, and translation/transcription/post-transcriptional modification. This study provides proteomic profiles that poplars responding to solar UV-B radiation, and it also provides new insights into differentially sex-related responses to UV-B radiation.
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Frontiers in plant science2017. Zhang, Yunxiang et al.
Chinese Acad Sci, Inst Mt Hazards & Environm, Key Lab Mt Surface Proc & Ecol Regulat, Chengdu, Peoples R China
ABSTRACT:With increasing altitude, solar UV-B radiation is enhanced. Based on the phenomenon of male-biased sex ratio of Populus cathayana Rehder in high altitude alpine area, we hypothesized that males have a faster and more sophisticated responsive mechanism to high UV-B radiation than that of females. Our previous studies have shown sexually different responses to high UV-B radiation were existed in P. cathayana at the morphological, physiological, and transcriptomic levels. However, the responses at the proteomic level remain unclear. In this study, an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteome analysis was performed in P. cathayana females and males. A total of 2,405 proteins were identified, with 331 proteins defined as differentially expressed proteins (DEPs). Among of these, 79 and 138 DEPs were decreased and 47 and 107 DEPs were increased under high solar UV-B radiation in females and males, respectively. A bioinformatics analysis categorized the common responsive proteins in the sexes as related to carbohydrate and energy metabolism, translation/transcription/post-transcriptional modification, photosynthesis, and redox reactions. The responsive proteins that showed differences in sex were mainly those involved in amino acid metabolism, stress response, and translation/transcription/post-transcriptional modification. This study provides proteomic profiles that poplars responding to solar UV-B radiation, and it also provides new insights into differentially sex-related responses to UV-B radiation.
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Analytical chemistry2017. Zhao, Qun et al.
Chinese Acad Sci, Dalian Inst Chem Phys, Natl Chromatog R&A Ctr, Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China
ABSTRACT:Although great achievement has been made in the mapping of human proteome, the efficiency of sample preparation still needs to be improved, especially for membrane proteins. Herein, we presented a novel method to deepen proteome coverage by the sequential extraction of proteins using urea and 1-dodecy1-3- methylimidazolium chloride (C12Im-Cl). With such a strategy, the commonly lost hydrophobic proteins by 8 M urea extraction could be further recovered by C12Im-Cl, as well as the suppression effect of high abundance soluble proteins could be decreased. Followed by the in situ sample preparation and separation with different stationary phases, more than 9810 gene products could be identified, covering 8 orders of magnitude in abundance, which was, to the best of our knowledge, the largest data set of HeLa cell proteome. Compared with previous work, not only the number of proteins identified was obviously increased, but also the analysis time was shortened to a few days. Therefore, we expect that such a strategy has great potential applications to achieve unprecedented coverage for proteome analysis.
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Nature Communications2017. Han, CF et al.
Ocean Univ China, Sch Med & Pharm, 5 Yushan Rd, Qingdao 266003, Peoples R China.
ABSTRACT:SIRT1 is the most evolutionarily conserved mammalian sirtuin, and it plays a vital role in the regulation of metabolism, stress responses, genome stability, and ageing. As a stress sensor, SIRT1 deacetylase activity is significantly increased during stresses, but the molecular mechanisms are not yet fully clear. Here, we show that SIRT1 is dynamically modified with O-GlcNAc at Ser 549 in its carboxy-terminal region, which directly increases its deacetylase activity both in vitro and in vivo. The O-GlcNAcylation of SIRT1 is elevated during genotoxic, oxidative, and metabolic stress stimuli in cellular and mouse models, thereby increasing SIRT1 deacetylase activity and protecting cells from stress-induced apoptosis. Our findings demonstrate a new mechanism for the activation of SIRT1 under stress conditions and suggest a novel potential therapeutic target for preventing age-related diseases and extending healthspan.
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Scientific reports2017. Zhang, Qi et al.
Philipps Univ Marburg, Dept Parasitol, Marburg, Germany
ABSTRACT:Malaria parasites modify their human host cell, the mature erythrocyte. This modification is mediated by a large number of parasite proteins that are exported to the host cell, and is also the underlying cause for the pathology caused by malaria infection. Amongst these proteins are many Hsp40 co-chaperones, and a single Hsp70. These proteins have been implicated in several processes in the host cell, including a potential role in protein transport, however the further molecular players in this process remain obscure. To address this, we have utilized chemical cross-linking followed by mass spectrometry and immunoblotting to isolate and characterize proteins complexes containing an exported Hsp40 (PFE55), and the only known exported Hsp70 (PfHsp70x). Our data reveal that both of these proteins are contained in high molecular weight protein complexes. These complexes are found both in the infected erythrocyte, and within the parasite-derived compartment referred to as the parasitophorous vacuole. Surprisingly, our data also reveal an association of PfHsp70x with components of PTEX, a putative protein translocon within the membrane of the parasitophorous vacuole. Our results suggest that the P. falciparum-infected human erythrocyte contains numerous high molecular weight protein complexes, which may potentially be involved in host cell modification.
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Molecular Biosystems2017. Karch, KR et al.
Univ Penn, Epigenet Inst, Perelman Sch Med, Dept Biochem & Mol Biophys, Philadelphia, PA 19104 USA.
ABSTRACT:ADP-ribosylation is a protein post-translational modification catalyzed by ADP-ribose transferases (ARTs). ART activity is critical in mediating many cellular processes, and is required for DNA damage repair. All five histone proteins are extensively ADP-ribosylated by ARTs upon induction of DNA damage. However, how these modifications aid in repair processes is largely unknown, primarily due to lack of knowledge about where they site-specifically occur on histones. Here, we conduct a comprehensive analysis of histone Asp/Glu ADP-ribosylation sites upon DNA damage induced by dimethyl sulfate (DMS). We also demonstrate that incubation of cell nuclei with NAD(+), as has been done previously in the literature, leads to spurious ADP-ribosylation levels of histone proteins. Altogether, we were able to identify 30 modification sites, 20 of which are novel. We also quantify the abundance of these modification sites during the course of DNA damage insult to identify which sites are critical for mediating repair. We found that every quantifiable site increases in abundance over time and that each identified ADP-ribosylation site is located on the surface of the nucleosome. Together, the data suggest specific Asp/Glu residues are unlikely to be critical for DNA damage repair and rather that this process is likely dependent on ADP-ribosylation of the nucleosomal surface in general.
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Journal of Proteome Research2017. Wang, YH et al.
Beijing Inst Radiat Med, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT:Although 5 years of the missing proteins (MPs) study have been completed, searching for MPs remains one of the core missions of the Chromosome-Centric Human Proteome Project (C-HPP). Following the next-50-MPs challenge of the C-HPP, we have focused on the testis-enriched MPs by various strategies since 2015. On the basis of the theoretical analysis of MPs (2017-01, neXtProt) using multiprotease digestion, we found that nonconventional proteases (e.g. LysargiNase, GluC) could improve the peptide diversity and sequence coverage compared with Trypsin. Therefore, a multiprotease strategy was used for searching more MPs in the same human testis tissues separated by 10% SDS-PAGE, followed by high resolution LC-MS/MS system (Q Exactive HF). A total of 7838 proteins were identified. Among them, three PE2 MPs in neXtProt 2017-01 have been identified: beta-defensin 123 (Q8N688, chr 20q), cancer/testis antigen family 45 member A10 (PODMU9, chr Xq), and Histone H2A-Bbd type 2/3 (P0C5Z0, chr Xq). However, because only one unique peptide of >= 9 AA was identified in beta-defensin 123 and Histone H2A-Bbd type 2/3, respectively, further analysis indicates that each falls under the exceptions clause of the HPP Guidelines v2.1. After a spectrum quality check, isobaric PTM and single amino acid variant (SAAV) filtering, and verification with a synthesized peptide, and based on overlapping peptides from different proteases, these three MPs should be considered as exemplary examples of MPs found by exceptional criteria. Other MPs were considered as candidates but need further validation. All MS data sets have been deposited to the ProteomeXchange with identifier PXD006465.
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Journal of Proteome Research2017. Peng, XH et al.
Wuhan Univ, Sch Pharmaceut Sci, Minist Educ, Key Lab Combinatorial Biosynth & Drug Discovery, Wuhan 430072, Peoples R China.
ABSTRACT:Identifying missing proteins (MPs) has been one of the critical missions of the Chromosome-Centric Human Proteome Project (C-HPP). Since 2012, over 30 research teams from 17 countries have been trying to search biochemical strategies. MPs mainly fall into the following adequate and accurate evidence of MPs through various classes: (1) low-molecular-weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), (4) nucleic acid associated proteins, (5) low abundance, and (6) unexpressed genes. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, and 8962 proteins were identified, including 6415 phosphoproteins, and 44 728 phosphorites, of which 10 266 were unreported previously. In total, 75 candidate detections were found, including 45 phoshoproteins. GO analysis for these 75 candidate detections revealed that these proteins mainly clustered as membrane proteins and took part in nephron and kidney development. After rigorous screening and manual check, 9 of them were verified with the synthesized peptides. Finally, only one missing protein was confirmed. All mass spectrometry data from this study have been deposited in the PRIDE with identifier PXD006482.
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Journal of Proteome Research2017. Yu, FC et al.
Hong Kong Univ Sci & Technol, Dept Elect & Comp Engn, Hong Kong, Hong Kong, Peoples R China.
ABSTRACT:Chemical cross-linking coupled to mass spectrometry is a powerful tool to study protein protein interactions and protein conformations. Two linked peptides are ionized and fragmented to produce a tandem mass spectrum. In such an experiment, a tandem mass spectrum contains ions from two peptides. The peptide identification problem becomes a peptide peptide pair identification problem. Currently, most tools do not search all possible pairs due to the quadratic time complexity. Consequently, missed findings are unavoidable. In our previous work, we developed a tool named ECL to search all pairs of peptides exhaustively. Unfortunately, it is very slow due to the quadratic computational complexity, especially when the database is large. Furthermore, ECL uses a score function without statistical calibration, while researchers1-3 have proposed that it is inappropriate to directly compare uncalibrated scores because different spectra have different random score distributions. Here we propose an advanced version of ECL, named ECL2. It achieves a linear time and space complexity by taking advantage of the additive property of a score function. It can search a data set containing tens of thousands of spectra against a database containing thousands of proteins in a few hours. Comparison with other five state-of-the-art tools shows that ECL2 is much faster than pLink, StavroX, ProteinProspector, and ECL. Kojak is the only one that is faster than ECL2, but Kojak does not exhaustively search all possible peptide pairs. The comparison shows that ECL2 has the highest sensitivity among the state-of-the-art tools. The experiment using a large-scale in vivo cross-linking data set demonstrates that ECL2 is the only tool that can find the peptide-spectrum matches (PSMs) passing the false discovery rate/q-value threshold. The result illustrates that the exhaustive search and a well-calibrated score function are useful to find PSMs from a huge search space.
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Journal of Proteome Research2017. Gashash, EA et al.
Georgia State Univ, Ctr Diagnost & Therapeut, Atlanta, GA 30302 USA.
ABSTRACT:Human plasma von Willebrand Factor (VWF) plays essential roles in primary hemostasis in cooperation with other coagulations factors. There is ample indication that glycosylation affects many biological phases during the protein life cycle. However, comprehensive characterization of all probable N-glycosites simultaneous with O-glycosites is still not fully revealed. Thus, the intention of this exploration was to estimate the occupancy of all canonical N-glycosites besides simultaneous characterization of N- and O-glycoforms. An RP-LC-MS/MS system functionalized with CID and HCD tandem mass was utilized to analyze VWF. N-Glycosite occupancy varied along the protein backbone chain. Out of 257 HCD spectra, 181 characterized glycoforms were specified as either N- or O-glycosites. Sequential cleavage of glycosidic bonds along with Human Database mass matching have confirmed the glycoform structures. A total of 173 glycoforms represented most commonly biantennary and infrequently tri- and tetra-antennary N-glycans beside high mannose, hybrid, ABH antigen-terminated, and sulfated N-glycans. Many glycoforms were common across all N-sites. Noteworthy, previously unreported N-glycosites within domain D'(TIL'-E') showed glycosylation. Moreover, sialylated core 1 and core 2 O-glycans were detected on 2298T. Given subtle characterization of site-specific glycoforms, we can attain a profound understanding of the biological roles of VWF as well as facilitate the production of VWF-based therapeutics.y
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