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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Analytical Chemistry2017. Tian, CP et al.
Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci, State Key Lab Prote, Beijing 102206, Peoples R China.
ABSTRACT:Proteins can undergo oxidative cleavage by in-vitro metal-catalyzed oxidation (MCO) in either the aamidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal alpha-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-alpha-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.
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Science Bulletin2017. Yang, KG et al.
Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Dalian 116023, Peoples R China.
ABSTRACT:The complications of hemodialysis accompanied the hemodialysis and threaten the patients' life. Besides the loss of nutrient substance, such as amino acid and vitamin, we found new clues that the adsorbed proteins on common-used polysulfone-based dialysis membrane might be the reason according to the qualitative proteomic study by ionic liquid assisted sample preparation method. Our results indicated that the adsorbed proteins on the membrane were related with complement activation, blood coagulation, and leukocyte-related biological process. The quantitative proteome further demonstrated some significant changes of signal proteins in the post-dialysis plasma after the hemodialysis, such as beta-2-microglobulin and platelet factor-4, which would further verify these new clues. (C) 2017 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.
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Proteomics2017. Li, YC et al.
Beijing Proteome Res Ctr, 38 Sci Pk Rd, Beijing 102206, Peoples R China.
ABSTRACT:Protein N-terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N-terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N-terminal peptide losses. In this study, a fast, one-step approach utilizing (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N-terminal peptide enrichment and analysis. Based on the characteristics of TMPP-derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision-induced dissociation or HCD fragmentation modes, first the SDS-PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP-modified N-terminal spectra. When utilizing a low amount of starting material (similar to 20 mu g protein), a total of 581 yeast N-terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N-terminomic studies.
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Science Translational Medicine2017. Kim, J et al.
Univ Penn, Inst Regenerat Med, Dept Cell & Dev Biol, Abramson Canc Ctr,Tumor Biol Program,Perelman Sch, 9-131 Smilow Ctr Translat Res,3400 Civ Ctr Blvd, Philadelphia, PA 19104 USA.
ABSTRACT:Markers are needed to facilitate early detection of pancreatic ductal adenocarcinoma (PDAC), which is often diagnosed too late for effective therapy. Starting with a PDAC cell reprogramming model that recapitulated the progression of human PDAC, we identified secreted proteins and tested a subset as potential markers of PDAC. We optimized an enzyme-linked immunosorbent assay (ELISA) using plasma samples from patients with various stages of PDAC, from individuals with benign pancreatic disease, and from healthy controls. A phase 1 discovery study (n = 20), a phase 2a validation study (n = 189), and a second phase 2b validation study (n = 537) revealed that concentrations of plasma thrombospondin-2 (THBS2) discriminated among all stages of PDAC consistently. The receiver operating characteristic (ROC) c-statistic was 0.76 in the phase 1 study, 0.84 in the phase 2a study, and 0.87 in the phase 2b study. The plasma concentration of THBS2 was able to discriminate resectable stage I cancer as readily as stage III/IV PDAC tumors. THBS2 plasma concentrations combined with those for CA19-9, a previously identified PDAC marker, yielded a c-statistic of 0.96 in the phase 2a study and 0.97 in the phase 2b study. THBS2 data improved the ability of CA19-9 to distinguish PDAC from pancreatitis. With a specificity of 98%, the combination of THBS2 and CA19-9 yielded a sensitivity of 87% for PDAC in the phase 2b study. A THBS2 and CA19-9 blood marker panel measured with a conventional ELISA may improve the detection of patients at high risk for PDAC.
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Talanta2017. Liu, LK et al.
457 Zhongshan Rd, Dalian 116023, Peoples R China.
ABSTRACT:Circulating tumor cells hold the key to predicting the prognosis and discovering the therapeutic targets. Herein, we proposed a strategy to develop an aptamer-immobilized open tubular capillary column by which SMMC-7721 human hepatoma cells (SMMC-7721 cells) could be captured with an over 70% of capture efficiency and a 3.0 +/- 0.2 of enrichment factor. Owing to the compatibility of the column, the captured cells by the column could be analyzed by LC-MS from protein level and 5 unique proteins of SMMC-7721 cells were identified which could be used as markers to identify SMMC-7721 cells when Jurkat T-leukemia cells (Jurkat cells) were employed as interfering cells. As the key component, the aptamer-immobilized column had the potential to be integrated into the platform for separating, enriching and characterizing rare cells simultaneously.
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Proteomics Clinical Applications2017. Han, S et al.
Chinese Acad Sci, Natl Chromatog Res & Anal Ctr, Dalian Inst Chem Phys, Key Lab Separat Sci Analyt Chem, Dalian, Peoples R China.
ABSTRACT:Hemodialysis is one of the most important therapies for patients with uremia, and the dialysis membrane is the predominant factor that impacts the efficiency of dialysis. Here, a protein adsorption on two different membranes is investigated to provide a basis for improving dialysis materials. Two cases treated with the Polyflux 14L low-flux dialyzer and the Polyflux 140H high-flux dialyzers during two continuous therapies are selected. Four used dialyzers from selected patients are infused with C12Im-Cl to elute the adsorbed proteins. Then labeled digested proteins adsorb by Polyflux 140H and Polyflux 14L with (CD2O)-C-13 and NaCNBD3 (light labeling, L) and CD2O and NaCNBH3 (heavy labeling, H), respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to identify the proteins. According to the ratio (Light labeling/Heavy labeling), the eluted proteins are divided into three groups: significantly higher, significantly lower, and no significant differences with a ratio of > 2, < 0.5, and 0.5-2, respectively. A total of 668 proteins are identified by LC-MS/MS, among which 177 proteins are retained more by the Polyflux 140H membrane (ratio > 2), 320 proteins are retained more by the Polyflux 14L membrane (ratio < 0.5), and 171 proteins show no significant difference (ratio 0.5-2) between the two types of membranes. Statistical significance is shown in the percentage of adsorbed proteins with an isoelectric point (pI) ranging from 9 to 10 (19.08 versus 7.69%; chi(2) = 11.87, p = 0.0006). Proteins with a molecular weight (MW) of 10-15 kDa tend to deposit on Polyflux 140H compared with Polyflux 14L (25 versus 9.23%; chi(2) = 18.66, p = 0.0000) and proteins with a MW of 30-60 kDa tend to deposit on Polyflux 14L compared with Polyflux 140H (36.54 versus 22.37%; chi(2) = 8.96, p = 0.0028). According to gene ontology analysis, the proteins adsorbed by dialysis membranes are closely related to activation of complement system and the coagulation cascade. The proteins adsorbed by Polyflux 140H and Polyflux 14L show significant differences in PI, MW, and protein class. Proteomic techniques are an effective approach for studying hemodialysis membranes.
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International Journal of Molecular Sciences2017. Xu, YR et al.
Natl Ctr Prot Sci Beijing, Natl Engn Res Ctr Prot Drugs, State Key Lab Prote, Beijing Proteome Res Ctr,Inst Radiat Med, Beijing 102206, Peoples R China.
ABSTRACT:Chronic hepatitis B virus (HBV) infection has been considered as the major cause of hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) has been reported to be oncogenic. The underlying mechanisms of HBV-related HCC are not fully understood, and the role played by the HBx protein in HBV induced carcinogenesis remains controversial. CDC42, a member of the Rho GTPase family, has been reported to be overexpressed in several different cancers, including HBV-related HCC. However, the specific role of CDC42 in HCC development remains unclear. Here, we investigated the cellular mechanisms by which CDC42 was responsible for the higher proliferation of HuH-7 cells mediated by HBx. We found that the expression level of CDC42 and its activity were significantly increased in HuH-7-HBx cells. The deficiency of CDC42 using the CRISPR/Cas9 system and inhibition by specific inhibitor CASIN led to the reduction of HBx-mediated proliferation. Furthermore, we observed that IQ Motif Containing GTPase Activating Protein 1 (IQGAP1), the downstream mediator of the CDC42 pathway, might be involved in the carcinogenesis induced by HBx. Therefore, the HBx/CDC42/IQGAP1 signaling pathway may potentially play an important role in HBx-mediated carcinogenesis.
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PNAS2017. Wu, Y et al.
Chinese Acad Sci, Key Lab Pathogen Microbiol & Immunol, Inst Microbiol, Beijing 100101, Peoples R China.
ABSTRACT:Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae. Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4-5 reveals that helices alpha 6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.
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Antioxidants & Redox Signaling2017. Mauney, CH et al.
Wake Forest Sch Med, Ctr Struct Biol, Dept Biochem, Winston Salem, NC 27157 USA.
ABSTRACT:Aims: Proliferative signaling involves reversible posttranslational oxidation of proteins. However, relatively few molecular targets of these modifications have been identified. We investigate the role of protein oxidation in regulation of SAMHD1 catalysis. Results: Here we report that SAMHD1 is a major target for redox regulation of nucleotide metabolism and cell cycle control. SAMHD1 is a triphosphate hydrolase, whose function involves regulation of deoxynucleotide triphosphate pools. We demonstrate that the redox state of SAMHD1 regulates its catalytic activity. We have identified three cysteine residues that constitute an intrachain disulfide bond redox switch that reversibly inhibits protein tetramerization and catalysis. We show that proliferative signals lead to SAMHD1 oxidation in cells and oxidized SAMHD1 is localized outside of the nucleus. Innovation and Conclusions: SAMHD1 catalytic activity is reversibly regulated by protein oxidation. These data identify a previously unknown mechanism for regulation of nucleotide metabolism by SAMHD1. Antioxid. Redox Signal. 27, 1317-1331.
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