pFind Studio: a computational solution for mass spectrometry-based proteomics

2017




Informed-Proteomics: open-source software package for top-down proteomics.
Nature Methods. 2017. Jungkap Park. et al. Pacific Northwest National Laboratory
ABSTRACT: Top-down proteomics, the analysis of intact proteins in their endogenous form, preserves valuable information about post-translation modifications, isoforms and proteolytic processing. The quality of top-down liquid chromatography–tandem MS (LC-MS/MS) data sets is rapidly increasing on account of advances in instrumentation and sample-processing protocols. However, top-down mass spectra are substantially more complex than conventional bottom-up data. New algorithms and software tools for confident proteoform identification and quantification are needed. Here we present Informed-Proteomics, an open-source software suite for top-down proteomics analysis that consists of an LC-MS feature-finding algorithm, a database search algorithm, and an interactive results viewer.
[more...]
Use: pTop



The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage.
Molecular BioSystems. 2017. Kelly R. Karch. et al. University of Pennsylvania
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.
[more...]
Use: pFind



Protein-Level Integration Strategy of Multiengine MS Spectra Search Results for Higher Confidence and Sequence Coverage.
Journal of Proteome Research. 2017. Panpan Zhao. et al. Jinan University
ABSTRACT: Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines. We used seven mainstream search engines (Andromeda, Mascot, OMSSA, X!Tandem, pFind, InsPecT, and ProVerB) to search the same label-free MS data sets of human cell lines Hep3B, MHCCLM3, and MHCC97H from the Chinese C-HPP Consortium for Chromosomes 1, 8, and 20.
[more...]
Use: pFind



A Chemoproteomic Platform To Assess Bioactivation Potential of Drugs.
Chemical Research in Toxicology. 2017. Rui Sun. et al. Beijing Proteome Research Center
ABSTRACT: Reactive metabolites (RM) formed from bioactivation of drugs can covalently modify liver proteins and cause mechanism-based inactivation of major cytochrome P450 (CYP450) enzymes. Risk of bioactivation of a test compound is routinely examined as part of lead optimization efforts in drug discovery. Here we described a chemoproteomic platform to assess in vitro and in vivo bioactivation potential of drugs. This platform enabled us to determine reactivity of thousands of proteomic cysteines toward RMs of diclofenac formed in human liver microsomes and living animals. We pinpointed numerous reactive cysteines as the targets of RMs of diclofenac, including the active (heme-binding) sites on several key CYP450 isoforms (1A2, 2E1 and 3A4 for human, 2C39 and 3A11 for mouse).
[more...]
Use: pFind



Proteomic study provides new clues for complications of hemodialysis caused by dialysis membrane.
Science Bulletin. 2017. Kaiguang Yang. et al. Dalian Institute of Chemical Physics
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.
Use: pFind



Identification of Missing Proteins in the Phosphoproteome of Kidney Cancer.
Journal of Proteome Research. 2017. Xuehui Peng. et al. Beijing Proteome Research Center
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 adequate and accurate evidence of MPs through various biochemical strategies. MPs mainly fall into the following 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 phosphosites, of which 10 266 were unreported previously.
[more...]
Use: pFind, pBuild



Structure of an Intron Lariat Spliceosome from Saccharomyces cerevisiae.
Cell. 2017. Ruixue Wan. et al. Tsinghua-Peking Joint Center for Life Sciences
ABSTRACT: The disassembly of the intron lariat spliceosome (ILS) marks the end of a splicing cycle. Here we report a cryoelectron microscopy structure of the ILS complex from Saccharomyces cerevisiae at an average resolution of 3.5 Å. The intron lariat remains bound in the spliceosome whereas the ligated exon is already dissociated. The step II splicing factors Prp17 and Prp18, along with Cwc21 and Cwc22 that stabilize the 5′ exon binding to loop I of U5 small nuclear RNA (snRNA), have been released from the active site assembly. The DEAH family ATPase/helicase Prp43 binds Syf1 at the periphery of the spliceosome, with its RNA-binding site close to the 3′ end of U6 snRNA.
[more...]
Use: pLink



Characterizing protein dynamics with integrative use of bulk and single-molecule techniques.
Biochemistry. 2017. Zhu Liu. et al. Wuhan Institute of Physics and Mathematics
ABSTRACT: A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques.
[more...]
Use: pLink



Conserved and unique features of the fission yeast core Atg1 complex.
Autophagy. 2017. Tamiza Nanji. et al. University of British Columbia
ABSTRACT: Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature.
[more...]
Use: pLink



Mechanism of Transcription Anti-termination in Human Mitochondria.
Cell. 2017. Hauke S. Hillen. et al. Max Planck Institute for Biophysical Chemistry
ABSTRACT: In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA—the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream “sliding clamp,” providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer.
[more...]
Use: pLink



Structure of RNA polymerase bound to ribosomal 30S subunit.
eLife. 2017. Gabriel Demo. et al. University of Massachusetts Medical School
ABSTRACT: In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome.
[more...]
Use: pLink



DISC: Disulfide Linkage Characterization from Tandem Mass Spectra.
Bioinformatics. 2017. Yi Liu. et al. University of Western Ontario
ABSTRACT: Enzymatic digestion under appropriate reducing conditions followed by mass spectrometry analysis has emerged as the primary method for disulfide bond analysis. The large amount of mass spectral data collected in the mass spectrometry experiment requires effective computational approaches to automate the interpretation process. Although different approaches have been developed for such purpose, they always choose to ignore the frequently observed internal ion fragments and they lack a reasonable quality control strategy and calibrated scoring scheme for the statistical validation and ranking of the reported results.In this research, we present a new computational approach, DISC (DISulfide bond Characterization), for matching an input MS/MS spectrum against the putative disulfide linkage structures hypothetically constructed from the protein database.
[more...]
Use: pLink-SS



Multi-Protease Strategy Identifies Three PE2 Missing Proteins in Human Testis Tissue.
Journal of Proteome Research. 2017. Yihao Wang. et al. Beijing Proteome Research Center
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.
[more...]
Use: pFind, pBuild, pLabel



An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly.
The EMBO Journal. 2017. Shrutee Jakhanwal. et al. Max Planck Institute for Biophysical Chemistry
ABSTRACT: Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1.
[more...]
Use: pLink



SUMO-Targeted DNA Translocase Rrp2 Protects the Genome from Top2-Induced DNA Damage.
Molecular Cell. 2017. Yi Wei. et al. National Institute of Biological Sciences
ABSTRACT: The action of DNA topoisomerase II (Top2) creates transient DNA breaks that are normally concealed inside Top2-DNA covalent complexes. Top2 poisons, including ubiquitously present natural compounds and clinically used anti-cancer drugs, trap Top2-DNA complexes. Here, we show that cells actively prevent Top2 degradation to avoid the exposure of concealed DNA breaks. A genome-wide screen revealed that fission yeast cells lacking Rrp2, an Snf2-family DNA translocase, are strongly sensitive to Top2 poisons. Loss of Rrp2 enhances SUMOylation-dependent ubiquitination and degradation of Top2, which in turn increases DNA damage at sites where Top2-DNA complexes are trapped. Rrp2 possesses SUMO-binding ability and prevents excessive Top2 degradation by competing against the SUMO-targeted ubiquitin ligase (STUbL) for SUMO chain binding and by displacing SUMOylated Top2 from DNA.
[more...]
Use: pLink



A new role for FBP21 as regulator of Brr2 helicase activity.
Nucleic Acids Research. 2017. Lisa M. Henning. et al. Freie Universität Berlin
ABSTRACT: Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity.
[more...]
Use: pLink



Structural basis for substrate selection by the translocation and assembly module of the β-barrel assembly machinery.
Molecular Microbiology. 2017. Rebecca S. Bamert. et al. Monash University
ABSTRACT: The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the β-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex. A peptide scan revealed that TamA and BamA bind the β-strands of FimD, and do so selectively.
[more...]
Use: pLink



Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation.
Autophagy. 2017. Jing-Xiang Zheng. et al. Tsinghua-Peking Joint Center for Life Sciences
ABSTRACT: PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45.
[more...]
Use: pLink



Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope.
Proceedings of the National Academy of Sciences of the United States of America. 2017. Yan Wu. et al. Institute of Microbiology, CAS
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.
[more...]
Use: pLink



Combining De Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics.
Journal of Proteome Research. 2017. Bernhard Blank-Landeshammer. et al. Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V.
ABSTRACT: Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms.
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Use: pNovo+



Impact of a priori MS/MS intensity distributions on database search for peptide identification.
Digital Signal Processing. 2017. Hatem Loukil. et al. University of Sfax
ABSTRACT: Many database search methods have been developed for peptide identification throughout a large peptide data set. Most of these approaches attempt to build a decision function that allows the identification of an experimental spectrum. This function is built either starting from similarity measures for the database peptides to identify the most similar one to a given spectrum, or by applying useful learning techniques considering the database itself as a training data. In this paper, we propose a peptide identification method based on a similarity measure for peptide-spectrum matches. Our method takes into account peak intensity distribution and applies it in a probabilistic scoring model to rank peptide matches.
[more...]
Use: pFind



The Null-Test for peptide identification algorithm in Shotgun proteomics.
Journal of Proteomics. 2017. Shu-Rong Zhang. et al. Dalian Institute of Chemical Physics
ABSTRACT: The present research proposed general evaluation strategy named Null-Test for peptide identification algorithm in Shotgun proteomics. The Null-Test method based on random matching can be utilized to check whether the algorithm has a tendency to make a mistake or has potential bugs, faultiness, errors etc., and to validate the reliability of the identification algorithm. Unfortunately, none of the five famous identification software could pass the most stringent Null-Test. PatternLab had good performance in both Null-Test and routine search by making a good control on the overfitting with sound design. The fuzzy logics based method presented as another candidate strategy could pass the Null-Test and has competitive efficiency in peptide identification.
[more...]
Use: pFind



A rapid and easy protein N-terminal profiling strategy using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip.
Proteomics. 2017. Yanchang Li. et al. Beijing Proteome Research Center
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.
[more...]
Use: pFind



An Insight into Glyco-Microheterogeneity of Plasma von Willebrand Factor by Mass Spectrometry.
Journal of Proteome Research. 2017. Ebtesam A. Gashash. et al. Georgia State University
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.
[more...]
Use: pFind



Proteomics Investigations into Serum Proteins Adsorbed by High-Flux and Low-Flux Dialysis Membranes.
Science Translational Medicine. 2017. Jungsun Kim. et al. University of Pennsylvania
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.
[more...]
Use: pFind



Aptamer-immobilized open tubular capillary column to capture circulating tumor cells for proteome analysis.
Talanta. 2017. Lukuan Liu. et al. Dalian Institute of Chemical Physics
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.
Use: pFind



Proteomics Investigations into Serum Proteins Adsorbed by High-Flux and Low-Flux Dialysis Membranes.
PROTEOMICS - Clinical Applications. 2017. Shuai Han. et al. Dalian Institute of Chemical Physics
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, we investigated protein adsorption on two different membranes 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 were selected. Four used dialyzers from selected patients were infused with C12Im-Cl to elute the adsorbed proteins. Then labeled digested proteins adsorbed by Polyflux 140H and Polyflux 14L with 13CD2O and NaCNBD3 (light labeling, L) and CD2O and NaCNBH3 (heavy labeling, H), respectively. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the proteins.
[more...]
Use: pFind



Exhaustively Identifying Cross-Linked Peptides with a Linear Computational Complexity.
Journal of Proteome Research. 2017. Fengchao Yu. et al. The Hong Kong University of Science and Technology
ABSTRACT: Chemical cross-linking coupled with 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 don't search all possible pairs due to the quadratic time complexity. Consequently, missed findings are unavoidable. In our earlier 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.
[more...]
Use: pLink, pFind



Association of IL-10-Regulating MicroRNAs in Peripheral Blood Mononuclear Cells with the Pathogenesis of Autoimmune Thyroid Disease.
Immunological Investigations. 2017. Yukina Takuse. et al. Osaka University Graduate School of Medicine
ABSTRACT: Interleukin (IL)-10 is known to suppress inflammation in autoimmune diseases. IL-10 can be regulated by miRNAs. To elucidate the involvement of miRNAs that regulate IL-10 expression with the pathogenesis of autoimmune thyroid disease (AITD), we examined the expression levels of hsa-miR-27a-3p, hsa-miR-98-5p, hsa-miR-106a-5p, and hsa-miR-223-3p in peripheral blood mononuclear cells (PBMCs) from 43 patients with Graves’ disease (GD), 38 patients with Hashimoto’s disease (HD), and 21 healthy volunteers. We evaluated the association between the expression levels of four miRNAs and intracellular expression of IL-10 in PBMCs from 11 healthy volunteers. We also genotyped MIR27A rs895819 G/A and MIR106A rs3747440 C/G polymorphisms, which may be related to the expression of these miRNAs in 141 patients with GD, 178 patients with HD, and 84 healthy volunteers.
[more...]
Use: pMatch



应用随机寡核苷酸文库修饰磁性颗粒提高血浆蛋白质的鉴定覆盖度.
色谱. 2017. 邓楠. et al. 中国科学院大连化学物理研究所
ABSTRACT: 基于随机寡核苷酸与蛋白质之间的离子、亲和、疏水、氢键等相互作用力及多种空间结构作用,发展了一种基于随机寡核苷酸文库作为配基的新型血浆样品处理方法。采用寡核苷酸文库修饰的磁性颗粒(MNP@ssDNA)材料,在生理缓冲体系条件下捕获血浆样品中的蛋白质。比较了两种洗脱体系的洗脱效果,并利用nano-RPLC-ESI-MS/MS对获得的蛋白质酶解液组分进行分析。结果表明,MNP@ssDNA材料处理后的血浆蛋白质鉴定数量提升了约29.5%,两种洗脱体系呈现良好的互补性(26.7%)。血浆中前10种高丰度蛋白质的谱图占有率从处理前的31.82%降低到21.31%(洗脱体系1)和26.20%(洗脱体系2)。在鉴定到的蛋白质中,丰度最低的蛋白质在血浆中的质量浓度约为0.29ng/mL,该蛋白质仅在MNP@ssDNA材料处理后被鉴定到。结果证明MNP@ssDNA策略不仅能有效降低血浆中高丰度蛋白质的丰度,也为低丰度蛋白质的深度挖掘提供了新的思路。
Use: pXtract



Structural determinants of Neosartorya fischeri antifungal protein (NFAP) for folding, stability and antifungal activity.
Scientific Reports. 2017. László Galgóczy. et al. Medical University of Innsbruck
ABSTRACT: The recent global challenges to prevent and treat fungal infections strongly demand for the development of new antifungal strategies. The structurally very similar cysteine-rich antifungal proteins from ascomycetes provide a feasible basis for designing new antifungal molecules. The main structural elements responsible for folding, stability and antifungal activity are not fully understood, although this is an essential prerequisite for rational protein design. In this study, we used the Neosartorya fischeri antifungal protein (NFAP) to investigate the role of the disulphide bridges, the hydrophobic core, and the N-terminal amino acids in the formation of a highly stable, folded, and antifungal active protein. NFAP and its mutants carrying cysteine deletion (NFAPΔC), hydrophobic core deletion (NFAPΔh), and N-terminal amino acids exchanges (NFAPΔN) were produced in Pichia pastoris.
[more...]
Use: pXtract



Acetylation and phosphorylation control both local and global stability of the chloroplast F1 ATP synthase.
Scientific Reports. 2017. Carla Schmidt. et al. University of Oxford
ABSTRACT: ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ε and δ subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the ε subunit in accord with extended and folded forms of the subunit.
[more...]
Use: pXtract



In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis.
Analytical Chemistry. 2017. Qun Zhao. et al. Dalian Institute of Chemical Physics
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-dodecyl-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.
[more...]
Use: pBuild



基于梯度提升决策树的肽碎片离子强度建模.
山东理工大学学报(自然科学版). 2017. 怀浩. et al. 山东理工大学
ABSTRACT: 为找到对蛋白质鉴定算法影响较大的肽碎片离子特征,以提高鉴定结果的正确率,在碎片离子特征与强度信息的基础上进行建模,构建预测模型.实验首先使用pFind对串联质谱数据鉴定,将鉴定结果过滤出需要的肽序列;然后计算出离子质荷比与离子特征值,通过匹配离子的质荷比获取离子强度信息;使用强度信息与离子特征值构建libsvm格式文件,使用XGBoost构建预测模型,其中使用了梯度提升决策树算法;最后使用构建完成的预测模型对蛋白质产生的肽序列做离子强度理论预测.实验结果表明模型所预测的肽序列离子强度与实验离子强度有着较高的相似度,同时分析预测模型可以从预测树中发现肽序列碎裂的规律,提取肽碎片离子中对强度值影响较大的离子特征.
Use: pFind



Hepatitis B Virus X Protein Stimulates Proliferation, Wound Closure and Inhibits Apoptosis of HuH-7 Cells via CDC42.
International Journal of Molecular Sciences. 2017. Yongru Xu. et al. Beijing Proteome Research Centre
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.
[more...]
Use: pFind



Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides.
Analytical and Bioanalytical Chemistry. 2017. Zixiang Yu. et al. Anhui Medical University
ABSTRACT: Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in 18O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC–MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools.
[more...]
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Phosphorylation of LSD1 by PLK1 promotes its chromatin release during mitosis.
Cell & Bioscience. 2017. Bin Peng. et al. Shenzhen University School of Medicine
ABSTRACT: Lysine-specific histone demethylase 1 (LSD1) modulates chromatin status through demethylation of H3K4 and H3K9. It has been demonstrated that LSD1 is hyperphosphorylated and dissociates from chromatin during mitosis. However, the molecular mechanism of LSD1 detachment is unknown. In this report, we found that polo-like kinase 1 (PLK1) directly interacted with LSD1 and phosphorylated LSD1 at Ser-126 . Nocodazole-induced metaphase arrest promoted release of LSD1 from chromatin, and the phosphorylation-defective mutant LSD1 (S126A) failed to dissociate from chromatin upon nocodazole treatment. Taken together, our findings demonstrate that phosphorylation of LSD1 at Ser-126 by PLK1 promotes its release from chromatin during mitosis.
Use: pLabel



Molecular architecture of the 90S small subunit pre-ribosome.
eLife. 2017. Qi Sun. et al. Institute of Biophysics, CAS
ABSTRACT: Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome.
[more...]
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Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.
Cell Reports. 2017. Payel Sen. et al. UT MD Anderson Cancer Center
ABSTRACT: The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF.
[more...]
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The SAMHD1 dNTP Triphosphohydrolase Is Controlled by a Redox Switch.
Antioxidants & Redox Signaling. 2017. Christopher H. Mauney. et al. Wake Forest School of Medicine
ABSTRACT: 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. 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.
[more...]
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Acetylation and phosphorylation control both local and global stability of the chloroplast F1 ATP synthase.
Scientific Reports . 2017. Carla Schmidt. et al. University of Oxford
ABSTRACT: ATP synthases (ATPases) are enzymes that produce ATP and control the pH in the cell or cellular compartments. While highly conserved over different species, ATPases are structurally well-characterised but the existence and functional significance of many post-translational modifications (PTMs) is not well understood. We combined a range of mass spectrometric techniques to unravel the location and extent of PTMs in the chloroplast ATP synthase (cATPase) purified from spinach leaves. We identified multiple phosphorylation and acetylation sites and found that both modifications stabilise binding of ε and δ subunits. Comparing cross-linking of naturally modified cATPase with the in vitro deacetylated enzyme revealed a major conformational change in the ε subunit in accord with extended and folded forms of the subunit. Locating modified residues within the catalytic head we found that phosphorylated and acetylated residues are primarily on α/β and β/α interfaces respectively.
[more...]
Use: pXtract, pLink



Proteome-wide mapping of endogenous SUMOylation sites in mouse testis.
Molecular & Cellular Proteomics. 2017. Lili Cai. et al. Fudan University
ABSTRACT: SUMOylation is a reversible post-translational modification involved in various critical biological processes. To date, there is limited approach for endogenous wild-type SUMO-modified peptides enrichment and SUMOylation sites identification. In this study, we generated a high-affinity SUMO1 antibody to facilitate the enrichment of endogenous SUMO1-modified peptides from Trypsin/Lys-C protease digestion. Following secondary Glu-C protease digestion, we identified 53 high-confidence SUMO1-modified sites from mouse testis by using high-resolution mass spectrometry. Bioinformatics analyses showed that SUMO1-modified proteins were enriched in transcription regulation and DNA repair.
[more...]
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Structural Insights of WHAMM's Interaction with Microtubules by Cryo-EM.
Journal of Molecular Biology. 2017. Tianyang Liu. et al. Tsinghua University
ABSTRACT: WASP homolog associated with actin, membranes, and microtubules (WHAMM) is a vertebrate protein functioning in membrane tubulation for intracellular membrane trafficking and specific organelle formation. Composed of multiple domains, WHAMM can bind to membrane and microtubule (MT) and promote actin polymerization nucleation. Previous work revealed that WHAMM's activity to promote actin nucleation is repressed upon binding to MTs. Here, we discovered that WHAMM interacts with αβ-tubulin through a small peptide motif within its MT-binding domain. We reconstructed a high-resolution structure of WHAMM's MT-binding motif (MBM) assembling around MTs using cryo-electron microscopy and verified it with chemical cross-linking and mass spectrometry analysis. We also detected a conformational switch of this motif between the non-MT-bound state and the MT-bound state.
[more...]
Use: pLink, pQuant



Preparation and application of silver nanoparticle-functionalized magnetic graphene oxide nanocomposites.
Nanoscale. 2017. Bo Jiang. et al. Dalian Institute of Chemical Physics
ABSTRACT: Although selective enrichment of glycopeptides from complex biological samples is indispensable for mass spectrometry (MS)-based glycoproteomics, it still remains a great challenge due to the low abundance of glycoproteins and suppression of non-glycopeptides. In this study, silver nanoparticle-functionalized magnetic graphene oxide nanocomposites (GO/Fe3O4/PEI/Ag) were synthesized. Silver nanoparticles were generated in situ on the surface of magnetic graphene oxide using polyethylenimine as a reducing and stabilizing agent. The resulting material was used as an adsorbent for selective enrichment of glycopeptides. GO/Fe3O4/PEI/Ag nanocomposites offered excellent enrichment ability, which was attributed to the synergistic effect of polyethylenimine and silver nanoparticles.
[more...]
Use: pBuild



SpotLight Proteomics: uncovering the hidden blood proteome improves diagnostic power of proteomics.
Scientific Reports. 2017. Susanna L. Lundström. et al. Karolinska Institutet
ABSTRACT: The human blood proteome is frequently assessed by protein abundance profiling using a combination of liquid chromatography and tandem mass spectrometry (LC-MS/MS). In traditional sequence database search, many good-quality MS/MS data remain unassigned. Here we uncover the hidden part of the blood proteome via novel SpotLight approach. This method combines de novo MS/MS sequencing of enriched antibodies and co-extracted proteins with subsequent label-free quantification of new and known peptides in both enriched and unfractionated samples. In a pilot study on differentiating early stages of Alzheimer's disease (AD) from Dementia with Lewy Bodies (DLB), on peptide level the hidden proteome contributed almost as much information to patient stratification as the apparent proteome. Intriguingly, many of the new peptide sequences are attributable to antibody variable regions, and are potentially indicative of disease etiology. When the hidden and apparent proteomes are combined, the accuracy of differentiating AD (n = 97) and DLB (n = 47) increased from ≈85% to ≈95%. The low added burden of SpotLight proteome analysis makes it attractive for use in clinical settings.
Use: pNovo+




2016




From structure to redox: the diverse functional roles of disulfides and implications in disease.
Proteomics. 2016. Tyler J. Bechtel. et al. Boston College
ABSTRACT: This review provides a comprehensive overview of the functional roles of disulfide bonds and their relevance to human disease. The critical roles of disulfide bonds in protein structure stabilization and redox regulation of protein activity are addressed. Disulfide bonds are essential to the structural stability of many proteins within the secretory pathway and can exist as intramolecular or inter-domain disulfides. The proper formation of these bonds often relies on folding chaperones and oxidases such as members of the protein disulfide isomerase (PDI) family. Many of the PDI family members catalyze disulfide-bond formation, reduction and isomerization through redox-active disulfides and perturbed PDI activity is characteristic of carcinomas and neurodegenerative diseases. In addition to catalytic function in oxidoreductases, redox-active disulfides are also found on a diverse array of cellular proteins and act to regulate protein activity and localization in response to oxidative changes in the local environment.
[more...]
Use: pLink-SS



ABRF Proteome Informatics Research Group (iPRG)2015 Study: Detection of differentially abundant proteins in label-free quantitative LC-MS/MS experiments.
Journal of proteome research. 2016. Meena Choi. et al. Northeastern University
ABSTRACT: Detection of differentially abundant proteins in label-free quantitative shotgun liquid chromatography–tandem mass spectrometry (LC–MS/MS) experiments requires a series of computational steps that identify and quantify LC–MS features. It also requires statistical analyses that distinguish systematic changes in abundance between conditions from artifacts of biological and technical variation. The 2015 study of the Proteome Informatics Research Group (iPRG) of the Association of Biomolecular Resource Facilities (ABRF) aimed to evaluate the effects of the statistical analysis on the accuracy of the results.
[more...]
Use: pFind



Global Post-translational Modification Discovery.
Journal of proteome research. 2016. Qiyao Li. et al. University of Wisconsin
ABSTRACT: A new global post-translational modification (PTM) discovery strategy, G-PTM-D, is described. A proteomics database containing UniProt-curated PTM information is supplemented with potential new modification types and sites discovered from a first-round search of mass spectrometry data with ultra-wide precursor mass tolerance. A second-round search employing the supplemented database conducted with standard narrow mass tolerances yields deep coverage and a rich variety of peptide modifications with high confidence in complex unenriched samples. The G-PTM-D strategy represents a major advance to the previously reported G-PTM strategy and provides a powerful new capability to the proteomics research community.
Use: pFind



Global Proteomics Analysis of Protein Lysine Methylation.
Current Protocols in Protein Science. 2016. Xing-Jun Cao. et al. University of Pennsylvania
ABSTRACT: Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large-scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry.
Use: pFind



Immunopurification and Mass Spectrometry Identifies Protein Phosphatase 2A (PP2A) and BIN2/GSK3 as Regulators of AKS Transcription Factors in Arabidopsis.
Molecular Plant. 2016. Shuo-Lei Bu. et al. Hebei Normal University
ABSTRACT: ABA induces the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1, AKS2, and AKS3). The unphosphorylated AKSs facilitate stomatal opening through promoting the transcription of genes encoding inwardly rectifying K+ channels (Takahashi et al., 2013). AKS1 and AKS3 are also regulators of flowering (Ito et al., 2012). However, the kinases and phosphatases that directly control the phosphorylation status of AKSs in vivo have not been fully characterized. Here, our proteomic analyses provide evidence supporting that AKSs are phosphorylated by GSK3 kinases and dephosphorylated by protein phosphatase 2A (PP2A).
[more...]
Use: pFind



Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.
Cell. 2016. Javier Fernandez-Martinez. et al. The Rockefeller University
ABSTRACT: The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC’s central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.
Use: pLink



Molecular architecture of the yeast Elongator complex reveals an unexpected asymmetric subunit arrangement.
EMBO reports. 2016. Dheva T Setiaputra. et al. The University of British Columbia
ABSTRACT: Elongator is a ~850 kDa protein complex involved in multiple processes from transcription to tRNA modification. Conserved from yeast to humans, Elongator is assembled from two copies of six unique subunits (Elp1 to Elp6). Despite the wealth of structural data on the individual subunits, the overall architecture and subunit organization of the full Elongator and the molecular mechanisms of how it exerts its multiple activities remain unclear. Using single‐particle electron microscopy (EM), we revealed that yeast Elongator adopts a bilobal architecture and an unexpected asymmetric subunit arrangement resulting from the hexameric Elp456 subassembly anchored to one of the two Elp123 lobes that form the structural scaffold. By integrating the EM data with available subunit crystal structures and restraints generated from cross‐linking coupled to mass spectrometry, we constructed a multiscale molecular model that showed the two Elp3, the main catalytic subunit, are located in two distinct environments. This work provides the first structural insights into Elongator and a framework to understand the molecular basis of its multifunctionality.
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Enrichment of cross-linked peptides using charge based fractional diagonal chromatography (ChaFRADIC).
Journal of Proteomics. 2016. Verena Tinnefeld. et al. Leibniz-Institut für Analytische Wissenschaften
ABSTRACT: Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. Here, we present here a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Based on two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible and independent of the cross-linking chemistry and cross-linker properties.
Use: pLink



Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes.
NATURE. 2016. Shan Wu. et al. Carnegie Mellon University
ABSTRACT: Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm1, 2. Hundreds of assembly factors, organized into sequential functional groups3, 4, facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs.
[more...]
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Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides.
Analytical and Bioanalytical Chemistry. 2016. He Zhu. et al. Georgia State University
ABSTRACT: N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment. This method integrated the advantages of Click Maltose and zwitterionic HILIC (ZIC-HILIC) and showed a relatively higher specificity for N-glycosylated peptides. This strategy was then applied to tryptic digests of normal human serum, followed by deglycosylation using peptide-N-glycosidase F (PNGase F) in H218O. Subsequent LC-MS/MS analysis allowed for the assignment of 219 N-glycosylation sites from 115 serum N-glycoproteins. This study provides an alternative approach for N-glycopeptide enrichment and the method employed is effective for large-scale N-glycosylation site identification. Graphical abstract Proposed mechanism of glycopeptides enrichment using DEAE-Sepharose.
Use: pFind, pBuild



Detection of busulfan adducts on proteins.
Rapid Communications in Mass Spectrometry. 2016. Acosta-Martin AE. et al. University of Geneva
ABSTRACT: Busulfan is a bifunctional alkyl sulfonate antineoplastic drug. This alkylating agent was described as forming covalent adducts on proteins. However, only limited data are available regarding the interaction of busulfan with proteins. Mass spectrometry and bioinformatics were used to identify busulfan adducts on human serum albumin and hemoglobin. Albumin and hemoglobin were incubated with busulfan or control compounds, digested with trypsin and analyzed by LC-MS/MS on a Thermo Fisher LTQ Orbitrap Velos Pro. MS data were used to generate spectral libraries of non-modified peptides and an open modification search was performed to identify potential adduct mass shifts and possible modification sites. Results were confirmed by a second database search including identified mass shifts and by visual inspection of annotated tandem mass spectra of adduct-carrying peptides.
[more...]
Use: pLabel



A Combinational Strategy upon RNA Sequencing and Peptidomics Unravels a Set of Novel Toxin Peptides in Scorpion Mesobuthus martensii.
Toxins. 2016. Ning Luan. et al. Kunming Institute of Zoology
ABSTRACT: Scorpion venom is deemed to contain many toxic peptides as an important source of natural compounds. Out of the two hundred proteins identified in Mesobuthus martensii (M. martensii), only a few peptide toxins have been found so far. Herein, a combinational approach based upon RNA sequencing and Liquid chromatography-mass spectrometry/mass spectrometry (LC MS/MS) was employed to explore the venom peptides in M. martensii. A total of 153 proteins were identified from the scorpion venom, 26 previously known and 127 newly identified. Of the novel toxins, 97 proteins exhibited sequence similarities to known toxins, and 30 were never reported. Combining peptidomic and transcriptomic analyses, the peptide sequence of BmKKx1 was reannotated and four disulfide bridges were confirmed within it. In light of the comparison of conservation and variety of toxin amino acid sequences, highly conserved and variable regions were perceived in 24 toxins that were parts of two sodium channel and two potassium channel toxins families. Taking all of this evidences together, the peptidomic analysis on M. martensii indeed identified numerous novel scorpion peptides, expanded our knowledge towards the venom diversity, and afforded a set of pharmaceutical candidates.
Use: pLabel



Structure of the voltage-gated calcium channel Cav1.1 at 3.6 Å resolution.
Nature. 2016. Jianping Wu. et al. Tsinghua University
ABSTRACT: The voltage-gated calcium (Cav) channels convert membrane electrical signals to intracellular Ca2+-mediated events. Among the ten subtypes of Cav channel in mammals, Cav1.1 is specified for the excitation-contraction coupling of skeletal muscles. Here we present the cryo-electron microscopy structure of the rabbit Cav1.1 complex at a nominal resolution of 3.6 Å. The inner gate of the ion-conductin Classification of the particles yielded two additional reconstructions that reveal pronounced displacement of β1a and adjacent elements in α1. The atomic model of the Cav1.1 complex establishes a foundation for mechanistic understanding of excitation-contraction coupling and provides a three-dimensional template for molecular interpretations of the functions and disease mechanisms of Cav and Nav c channels.
Use: pParse, pLink



Xilmass: a new approach towards the identification of cross-linked peptides.
Analytical Chemistry. 2016. Şule Yılmaz. et al. Ghent University
ABSTRACT: Chemical cross-linking coupled with mass spectrometry plays an important role in unravelling protein interactions, especially weak and transient ones. Moreover, cross-linking complements several structural determination approaches such as cryo-EM. Although several computational approaches are available for the annotation of spectra obtained from cross-linked peptides, there remains room for improvement. Here, we present Xilmass, a novel algorithm to identify cross-linked peptides that introduces two new concepts: (i) the cross-linked peptides are represented in the search database such that the cross-linking sites are explicitly encoded, and (ii) the scoring function derived from the Andromeda algorithm was adapted to score against a theoretical tandem mass spectrometry (MS/MS) spectrum that contains the peaks from all possible fragment ions of a cross-linked peptide pair.
[more...]
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The Architecture of Trypanosoma brucei editosomes.
PNAS. 2016. Suzanne M. McDermott. et al. Center for Infectious Disease Research
ABSTRACT: Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra- and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies.
[more...]
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Pex17p-dependent assembly of Pex14p/Dyn2psubcomplexes of the peroxisomal protein import machinery.
European Journal of Cell Biology. 2016. Anna Chan. et al. University of Freiburg
ABSTRACT: Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p an
[more...]
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Peptide de novo sequencing of mixture tandem mass spectra.
Proteomics. 2016. Vladimir Gorshkov. et al. University of Southern Denmark Odense M
ABSTRACT: The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides.
[more...]
Use: pNovo



Structural characterization of coatomer in its cytosolic state.
Protein& Cell. 2016. Shengliu Wang. et al. Institute of Biophysics, Chinese Academy of Sciences
ABSTRACT: Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form.
[more...]
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Phosphoproteome Characterization of Human Colorectal Cancer SW620 Cell-Derived Exosomes and New Phosphosite Discovery for C-HPP.
Journal of Proteome Research. 2016. Jiahui Guo. et al. Jinan University
ABSTRACT: Identification of all phosphorylation forms of known proteins is a major goal of the Chromosome-Centric Human Proteome Project (C-HPP). Recent studies have found that certain phosphoproteins can be encapsulated in exosomes and function as key regulators in tumor microenvironment, but no deep coverage phosphoproteome of human exosomes has been reported to date, which makes the exosome a potential source for the new phosphosite discovery. In this study, we performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected.
[more...]
Use: pLabel



Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.
PLOS Pathogens. 2016. Dhana G. Gorasia. et al. The University of Melbourne
ABSTRACT: The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein.
[more...]
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Synthesis of zwitterionic polymer particles via combined distillation precipitation polymerization and click chemistry for high efficient enrichment of glycopeptide.
ACS Appl. Mater. Interfaces. 2016. Jianxi Liu. et al. Dalian Institute of Chemical Physics
ABSTRACT: Because of the low abundance of glycopeptide in natural biological samples, methods for efficient and selective enrichment of glycopeptides play a significant role in mass spectrometry (MS)-based glycoproteomics. In this study, a novel kind of zwitterionic hydrophilic interaction chromatography polymer particles, namely, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@l-Cys (poly(MBAAm-co-MAA)@l-Cys), for the enrichment of glycopeptides was synthesized by a facile and efficient approach that combined distillation precipitation polymerization (DPP) and “thiol–ene” click reaction. In the DPP approach, residual vinyl groups explored outside the core with high density, then the functional ligand cysteine was immobilized onto the surface of core particles by highly efficient thiol–ene click reaction. Taking advantage of the unique structure of poly(MBAAm-co-MAA)@l-Cys, the resulting particles possess remarkable enrichment selectivity for glycopeptides from the tryptic digested human immunoglobulin G.
[more...]
Use: pBuild



Deep Coverage Proteomics Identifies More Low-Abundance Missing Proteins in Human Testis Tissue with Q-Exactive HF Mass Spectrometer.
Journal of Proteome Research<. 2016. Wei Wei. et al. Beijing Proteome Research Center
ABSTRACT: Since 2012, missing proteins (MPs) investigation has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) through various biochemical strategies. On the basis of our previous testis MPs study, faster scanning and higher resolution mass-spectrometry-based proteomics might be conducive to MPs exploration, especially for low-abundance proteins. In this study, Q-Exactive HF (HF) was used to survey proteins from the same testis tissues separated by two separating methods (tricine- and glycine-SDS-PAGE), as previously described. A total of 8526 proteins were identified, of which more low-abundance proteins were uniquely detected in HF data but not in our previous LTQ Orbitrap Velos (Velos) reanalysis data. Further transcriptomics analysis showed that these uniquely identified proteins by HF also had lower expression at the mRNA level. Of the 81 total identified MPs, 74 and 39 proteins were listed as MPs in HF and Velos data sets, respectively.
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Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process.
Journal of Proteome Research. 2016. Mingzhi Zhao. et al. Beijing Proteome Research Center
ABSTRACT: A membrane protein enrichment method composed of ultracentrifugation and detergent-based extraction was first developed based on MCF7 cell line. Then, in-solution digestion with detergents and eFASP (enhanced filter-aided sample preparation) with detergents were compared with the time-consuming in-gel digestion method. Among the in-solution digestion strategies, the eFASP combined with RapiGest identified 1125 membrane proteins. Similarly, the eFASP combined with sodium deoxycholate identified 1069 membrane proteins; however, the in-gel digestion characterized 1091 membrane proteins. Totally, with the five digestion methods, 1390 membrane proteins were identified with ≥1 unique peptides, among which 1345 membrane proteins contain unique peptides ≥2.
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De novo Peptide Sequencing using CID and HCD Spectra Pairs.
Proteomics. 2016. Yan Yan. et al. University of Saskatchewan Saskatoon
ABSTRACT: In tandem mass spectrometry (MS/MS), there are several different fragmentation techniques possible including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), and electron transfer dissociation (ETD). When using pairs of spectra for de novo peptide sequencing, the most popular methods are designed for CID (or HCD) and ECD (or ETD) spectra because of the complementarity between them. Less attention has been paid to the use of CID and HCD spectra pairs. In this study, a new de novo peptide sequencing method is proposed for these spectra pairs. This method includes a CID and HCD spectra merging criterion and a parent mass correction step, along with improvements to our previously proposed algorithm for sequencing merged spectra.
[more...]
Use: pNovo



UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly.
Nature Communications. 2016. Mirjam Hunziker. et al. The Rockefeller University
ABSTRACT: Early eukaryotic ribosome biogenesis involves large multi-protein complexes, which co-transcriptionally associate with pre-ribosomal RNA to form the small subunit processome. The precise mechanisms by which two of the largest multi-protein complexes—UtpA and UtpB—interact with nascent pre-ribosomal RNA are poorly understood. Here, we combined biochemical and structural biology approaches with ensembles of RNA–protein cross-linking data to elucidate the essential functions of both complexes. We show that UtpA contains a large composite RNA-binding site and captures the 5′ end of pre-ribosomal RNA. UtpB forms an extended structure that binds early pre-ribosomal intermediates in close proximity to architectural sites such as an RNA duplex formed by the 5′ ETS and U3 snoRNA as well as the 3′ boundary of the 18S rRNA. Both complexes therefore act as vital RNA chaperones to initiate eukaryotic ribosome assembly.
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The anti-sigma factor RsrA responds to oxidative stress by reburying its hydrophobic core.
Nature Communications. 2016. Karthik V. Rajasekar. et al. University of Oxford
ABSTRACT: Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor σR preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA–σR complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA’s three zinc-binding cysteines, precipitates structural collapse to a compact state where all σR-binding residues are sequestered back into its hydrophobic core, releasing σR to activate transcription of anti-oxidant genes.
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Improvement of core-fucosylated glycoproteome coverage via alternating HCD and ETD fragmentation.
Journal of Proteomics. 2016. Cheng Ma. et al. Georgia State University
ABSTRACT: Core-fucosylation (CF) plays important roles in regulating biological processes in eukaryotes. Alterations of CF-glycosites or CF-glycans in bodily fluids correlate with cancer development. Therefore, global research of protein core-fucosylation with an emphasis on proteomics can explain pathogenic and metastasis mechanisms and aid in the discovery of new potential biomarkers for early clinical diagnosis. In this study, a precise and high throughput method was established to identify CF-glycosites from human plasma. We found that alternating HCD and ETD fragmentation (AHEF) can provide a complementary method to discover CF-glycosites. A total of 407 CF-glycosites among 267 CF-glycoproteins were identified in a mixed sample made from six normal human plasma samples. Among the 407 CF-glycosites, 10 are without the N-X-S/T/C consensus motif, representing 2.5% of the total number identified. All identified CF-glycopeptide results from HCD and ETD fragmentation were filtered with neutral loss peaks and characteristic ions of GlcNAc from HCD spectra, which assured the credibility of the results. This study provides an effective method for CF-glycosites identification and a valuable biomarker reference for clinical research.
[more...]
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The dynamic interactome and genomic targets of Polycomb complexes during stem-cell differentiation.
NATURE STRUCTURAL & MOLECULAR BIOLOGY. 2016. Susan L Kloet. et al. Radboud University Nijmegen
ABSTRACT: Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites.
[more...]
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Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.
NATURE COMMUNICATIONS. 2016. Xiangdong Zheng. et al. Tsinghua University.
ABSTRACT: Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule’s α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules.
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ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry.
Analytical and Bioanalytical Chemistry. 2016. Yurong Wen. et al. Ghent University
ABSTRACT: Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response.
[more...]
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The MTA1 subunit of the nucleosome remodeling and deacetylase complex can recruit two copies of RBBP4/7.
Protein Science. 2016. Jason W. Schmidberger. et al.  University of Sydney
ABSTRACT: The Nucleosome Remodeling and Deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4.
[more...]
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CryoEM structure of yeast cytoplasmic exosome complex.
Cell research. 2016. Jun-Jie Liu. et al.  Tsinghua University
ABSTRACT: The eukaryotic multi-subunit RNA exosome complex plays crucial roles in 3′-to-5′ RNA processing and decay. Rrp6 and Ski7 are the major cofactors for the nuclear and cytoplasmic exosomes, respectively. In the cytoplasm, Ski7 helps the exosome to target mRNAs for degradation and turnover via a through-core pathway. However, the interaction between Ski7 and the exosome complex has remained unclear. The transaction of RNA substrates within the exosome is also elusive.
[more...]
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A novel quantitative mass spectrometry platform for determining protein O-GlcNAcylation dynamics.
Molecular & Cellular Proteomics. 2016. Xiaoshi Wang. et al.  University of Pennsylvania School of Medicine, United States
ABSTRACT: Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing 13C-labeled UDP-GlcNAc from 13C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates.
[more...]
Use: pXtract, pParse, pFind



Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/Rpb1 rather than XPB or p44.
Molecular Cancer Therapeutics. 2016. Jun-Mei Yi. et al.  Shanghai Institute of Materia Medica
ABSTRACT: Multidrug resistance (MDR) is a major cause of tumor treatment failure; therefore, drugs that can avoid this outcome are urgently needed. We studied triptolide which directly kills MDR tumor cells with a high potency and a broad spectrum of cell death. Triptolide did not inhibit P-glycoprotein (P-gp) drug-efflux and reduced P-gp and mdr1 mRNA resulted from transcription inhibition. Transcription factors including c-Myc, SOX-2, OCT-4, and NANOG were not correlated with triptolide-induced cell killing but Rpb1, the largest subunit of RNA polymerase II, was critical in mediating triptolide's inhibition of MDR cells.
[more...]
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O-GlcNAcylation Antagonizes Phosphorylation of Cdh1 (CDC20 homologue 1).
The Journal of Biological Chemistry. 2016. Jie Tian. et al.  Capital Normal University
ABSTRACT: The anaphase promoting complex/cyclosome (APC/C) orchestrates various aspects of the eukaryotic cell cycle. One of its co-activators, Cdh1, is subject to myriad post-translational modifications, such as phosphorylation and ubiquitination. Herein, we identify the O-linked N-acetylglucosamine (O-GlcNAc) modification that occurs on Cdh1. Cdh1 is O-GlcNAcylated in cultured cells and mouse brain extracts. Mass spectrometry identifies an O-GlcNAcylated peptide that neighbors a known phosphorylation site. Cell synchronization and mutation studies reveal that O-GlcNAcylation of Cdh1 may antagonize its phosphorylation.
[more...]
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Increasing the Depth of Mass Spectrometry-based Structural Analysis of Protein Complexes through the Use of ultiple Cross-linkers.
Analytical Chemistry. 2016. Yuehe Ding. et al.  National Institute of Biological Sciences
ABSTRACT: Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS3 and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins. In order to find the cross-linkers that can best complement the performance of BS3 and DSS, we tested seven additional cross-linkers that either have different spacer arm structures or that target different amino acids (BS2G, EGS, AMAS, GMBS, Sulfo-GMBS, EDC, and TFCS).
[more...]
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Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states.
eLife. 2016. Dan Tan. et al.  National Institute of Biological Sciences
ABSTRACT: To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy.
[more...]
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Characterization of post-translational modifications in full-length human BMP-1 confirms the presence of a rare vicinal disulfide linkage in the catalytic domain and highlights novel features of the EGF domain.
Journal of Proteomics. 2016. Chien-Wen Hung. et al.  Christian-Albrechts-Universität zu Kiel
ABSTRACT: Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C185-C186 could be unambiguously identified.
[more...]
Use: pLink-SS



Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells.
Cancer Research. 2016. Chih-Hang Anthony Tang. et al.  The Wistar Institute
ABSTRACT: Endoplasmic reticulum (ER) stress responses through the IRE-1/XBP-1 pathway are required for the function of STING (TMEM173), an ER-resident transmembrane protein critical for cytoplasmic DNA sensing, IFN production, and cancer control. Here we show that the IRE-1/XBP-1 pathway functions downstream of STING and that STING agonists selectively trigger mitochondria-mediated apoptosis in normal and malignant B cells. Upon stimulation, STING was degraded less efficiently in B cells, implying that prolonged activation of STING can lead to apoptosis.
[more...]
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A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses.
Journal of Proteomics. 2016. Yehui Xiong. et al. Institute of Plant Protection
ABSTRACT: Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in the gene transcription regulation. Here, we report high quality proteome-scale data for lysine-acetylation (Kac) sites and Kac proteins in rice (Oryza sativa). A total of 1337 Kac sites in 716 Kac proteins with diverse biological functions and subcellular localizations were identified in rice seedlings. About 42% of the sites were predicted to be localized in the chloroplast. Seven putative acetylation motifs were detected. Phenylalanine, located in both the upstream and downstream of the Kac sites, is the most conserved amino acid surrounding the regions.
[more...]
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An NGS-Independent Strategy for Proteome-Wide Identification of Single Amino Acid Polymorphisms by Mass Spectrometry.
Analytical Chemistry. 2016. Yun Xiong. et al. Tianjin Institute of Industrial Biotechnology
ABSTRACT: Detection of proteins containing single amino acid polymorphisms (SAPs) encoded by nonsynonymous SNPs (nsSNPs) can aid researchers in studying the functional significance of protein variants. Most proteogenomic approaches for large-scale SAPs mapping require construction of a sample-specific database containing protein variants predicted from the next-generation sequencing (NGS) data. Searching shotgun proteomic data sets against these NGS-derived databases allowed for identification of SAP peptides, thus validating the proteome-level sequence variation.
[more...]
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GALNT14 genotype effectively predicts the therapeutic response in unresectable hepatocellular carcinoma treated with transcatheter arterial chemoembolization.
Pharmacogenomics. 2016. Kung-Hao Liang. et al. Chang Gung Memorial Hospital
ABSTRACT: Transcatheter arterial chemoembolization is currently the standard treatment in hepatocellular carcinoma patients with Barcelona Clinic Liver Cancer stage B. Genomic variants of GALNT14 were recently identified as effective predictors for chemotherapy responses in Barcelona Clinic Liver Cancer stage C patients.We investigated the prognosis predictive value of GALNT14 genotypes in 327 hepatocelluar carcinoma patients treated by transcatheter arterial chemoembolization.
[more...]
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Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics
ELECTROPHORESIS. 2016. Shanshan Li. et al. Nankai University
ABSTRACT: O-linked β-N-acetylglucosamine (O-GlcNAc) is emerging as an essential protein posttranslational modification in a range of organisms. It is involved in various cellular processes such as nutrient sensing, protein degradation, gene expression and is associated with many human diseases. Despite its importance, identifying O-GlcNAcylated proteins is a major challenge in proteomics. Here, using peracetylated N-azidoacetylglucosamine (Ac4 GlcNAz) as a bioorthogonal chemical handle, we described a gel-based mass spectrometry method for the identification of proteins with O-GlcNAc modification in A549 cells.
[more...]
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Quantitative profiling of the activity of protein lysine methyltransferase SMYD2 using SILAC-based proteomics.
MOLECULAR & CELLULAR POTEOMICS. 2016. Jonathan B. Olsen. et al. Lilly Research Laboratories
ABSTRACT: The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell.
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Application of de Novo Sequencing to Large-Scale Complex.
Journal of Proteome Research. 2016. Arun Devabhaktuni. et al. Stanford University
ABSTRACT: Dependent on concise, predefined protein sequence databases, traditional search algorithms perform poorly when analyzing mass spectra derived from wholly uncharacterized protein products. Conversely, de novo peptide sequencing algorithms can interpret mass spectra without relying on reference databases. However, such algorithms have been difficult to apply to complex protein mixtures, in part due to a lack of methods for automatically validating de novo sequencing results.
[more...]
Use: pNovo




2015




Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes.
Nucleic Acids Research. 2015. Christian Trahan. et al.  Université de Montréal
ABSTRACT: Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs.
[more...]
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Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.
Journal of Proteome Research. 2015. Shaohang Xu. et al.  BGI-Shenzhen
ABSTRACT: Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins.
[more...]
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Extracting Accurate Precursor Information for Tandem Mass Spectra by RawConverter.
Analytical Chemistry. 2015. Lin He. et al.  The Scripps Research Institute
ABSTRACT: Extraction of data from the proprietary RAW files generated by Thermo Fisher mass spectrometers is the primary step for subsequent data analysis. High resolution and high mass accuracy data obtained by state-of-the-art mass spectrometers (e.g., Orbitraps) can significantly improve both peptide/protein identification and quantification. We developed RawConverter, a stand-alone software tool, to improve data extraction on RAW files from high-resolution Thermo Fisher mass spectrometers.
[more...]
Use: pXtract



Gold nanoparticles immobilized hydrophilic monoliths with variable functional modification for highly selective enrichment and on-line deglycosylation of glycopeptides.
Analytica Chimica Acta. 2015. Yu Liang. et al.  Dalian Institute of Chemical Physics
ABSTRACT: The poly (glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monoliths modified with gold nanoparticles, with advantages of enhanced reactive sites, good hydrophilicity and facile modification, were prepared as the matrix, followed by variable functionalization with cysteine and PNGase F for glycopeptide enrichment and on-line deglycosylation respectively. By the cysteine functionalized monolithic column, glycopeptides could be efficiently and selectively enriched with good reproducibility based on hydrophilic interaction chromatography (HILIC). Furthermore, the enrichment was specially achieved in weak alkaline environment, with 10 mM NH4HCO3 as the elution buffer, compatible with deglycosylation conditions.
[more...]
Use: pBuild



Monitoring cellular phosphorylation signaling pathways into chromatin and down to the gene level.
MOLECULAR & CELLULAR POTEOMICS. 2015. Yumiao Han. et al. University of Pennsylvania School of Medicine
ABSTRACT: Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood.
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Proteomic analysis of Allium cepa var. agrogarum L. roots under copper stress.
Plant Soil. 2015. Rong Qin. et al. South China Normal University
ABSTRACT: In the present study, the effects of Cu (2.0 and 8.0 μM) on root growth of Allium cepa var. agrogarum L. were addressed and protein abundance levels were analyzed using the technology of proteomics combined with transcriptomics, in order to go deeper into the understanding of the mechanism of Cu toxicity on plant root systems at the protein level and to provide valuable information for monitoring and forecasting the effects of exposure to Cu in real scenarios conditions.
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Subset of Kappa and Lambda Germline Sequences Result in Light Chains with a Higher Molecular Mass Phenotype.
Journal of Proteome Research. 2015. David R. Barnidge. et al. Karolinska Institutet
ABSTRACT: In our previous work, we showed that electrospray ionization of intact polyclonal kappa and lambda light chains isolated from normal serum generates two distinct, Gaussian-shaped, molecular mass distributions representing the light-chain repertoire. During the analysis of a large (>100) patient sample set, we noticed a low-intensity molecular mass distribution with a mean of approximately 24 250 Da, roughly 800 Da higher than the mean of the typical kappa molecular-mass distribution mean of 23 450 Da. We also observed distinct clones in this region that did not appear to contain any typical post-translational modifications that would account for such a large mass shift.
[more...]
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The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90.
Scientific Reports. 2015. Veronika Haslbeck. et al. Technische Universität München
ABSTRACT: Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes.
[more...]
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Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification.
Molecular Cellular Proteomics. 2015. Matthew M Makowski. et al. Radboud University Nijmegen
ABSTRACT: In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis.
[more...]
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Experimental Validation of Bacillus anthracis A16R Proteogenomics.
Scientific Reports. 2015. Zhiqi Gao. et al. Beijing Institute of Biotechnology
ABSTRACT: Anthrax, caused by the pathogenic bacterium Bacillus anthracis, is a zoonosis that causes serious disease and is of significant concern as a biological warfare agent. Validating annotated genes and reannotating misannotated genes are important to understand its biology and mechanisms of pathogenicity. Proteomics studies are, to date, the best method for verifying and improving current annotations. To this end, the proteome of B. anthracis A16R was analyzed via one-dimensional gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
[more...]
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Probing the missing human proteome: A computational perspective.
Journal of Proteome Research. 2015. Dhirendra Kumar. et al. CSIR-Institute of Genomics and Integrative Biology
ABSTRACT: The missing human proteome comprises predicted protein-coding genes with no credible protein level evidence detected so far and constitutes ∼18% of the human protein coding genes (neXtProt release 19/9/2014). The missing proteins may be of pharmacological interest as many of these are membrane receptors, thus requiring comprehensive characterization. In the present study, we explored various computational parameters, crucial during protein searches from tandem mass spectrometry (MS) data, for their impact on missing protein identification.
[more...]
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一种结合生物医学知识的蛋白质组非标记定量分析方法及其应用.
生物化学与生物物理进展. 2015. 潘超. et al. 浙江大学生物医学工程与仪器科学学院
摘要:基于质谱的非标记定量方法能够对复杂蛋白质组进行规模化分析,同时,在定量分析的基础上理解和解释蛋白质组的功能和相互作用关系更有意义.这要建立一种有效的兼容定量和定性分析结果的方法.针对这一需求,本文首先借鉴了NSAF(normalized spectral abundance factor)算法采用肽段计数对蛋白质组数据进行定量,进一步结合共享肽对该方法进行优化.以此为基础,通过 g:Profiler 获取海量蛋白质组的功能注释信息,在定量分析的过程中,同步实现了对蛋白质组数据的功能性分析. 本文选择来自人心脏、 小鼠心脏、 小鼠肝脏的三组线粒体蛋白质组数据对该方法进行验证, 按照功能性分析将三组数据划分为若干功能组或信号通路, 并进行相关性、 功能聚类以及电子传递链分析. 结果表明,结合共享肽的优化算法克服了对低丰度蛋白质的错误估计, 提高了非标记定量的准确性. 同时,结合生物医学知识的分析方法解释了蛋白质组的功能和相互作用关系, 为差异比较蛋白质组学、疾病蛋白质组学以及功能蛋白质组学等组学研究提供了新的方法.
Use: pFind



Unique diversity of the venom peptides from the scorpion Androctonus bicolor revealed by transcriptomic and proteomic analysis.
Journal of Proteomics Research. 2015. Lei Zhang. et al. China University of Geosciences (Wuhan)
ABSTRACT: Androctonus bicolor is one of the most poisonous scorpion species in the world. However, little has been known about the venom composition of the scorpion. To better understand the molecular diversity and medical significance of the venom from the scorpion, we systematically analyzed the venom components by combining transcriptomic and proteomic surveys.
[more...]
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Towards elucidating the stability, dynamics and architecture of the nucleosome remodeling and deacetylase complex by using quantitative interaction proteomics.
The Federation of European Biochemical Societies Journal. 2015. Susan L. et al. Radboud University Nijmegen
ABSTRACT: The nucleosome remodeling and deacetylase (NuRD) complex is an evolutionarily conserved chromatin-associated protein complex. Although the subunit composition of the mammalian complex is fairly well characterized, less is known about the stability and dynamics of these interactions. Furthermore, detailed information regarding protein–protein interaction surfaces within the complex is still largely lacking.
[more...]
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The Tissue-Based Proteogenomics Reveals that Human Testis Endows Plentiful Missing Proteins.
Journal of Proteome Research. 2015. Yao Zhang. et al. Beijing Institute of Genomics
ABSTRACT: Investigations of missing proteins (MPs) are being endorsed by many bioanalytical strategies. We proposed that proteogenomics of testis tissue was a feasible approach to identify more MPs because testis tissues have higher gene expression levels. Here we combined proteomics and transcriptomics to survey gene expression in human testis tissues from three post-mortem individuals.
[more...]
Use: pLabel



The RNA helicase Aquarius exhibits structural adaptations mediating its recruitment to spliceosomes.
NATURE STRUCTURAL & MOLECULAR BIOLOGY. 2015. Inessa De. et al. Max Planck Institute
ABSTRACT: ​Aquarius is a multifunctional putative RNA helicase that binds precursor-mRNA introns at a defined position. Here we report the crystal structure of human ​Aquarius, revealing a central RNA helicase core and several unique accessory domains, including an ARM-repeat domain. We show that ​Aquarius is integrated into spliceosomes as part of a pentameric intron-binding complex (IBC) that, together with the ARM domain, cross-links to U2 snRNP proteins within activated spliceosomes; this suggests that the latter aid in positioning ​Aquarius on the intron. ​
[more...]
Use: pLink



Structure of a yeast spliceosome at 3.6-angstrom resolution.
Science. 2015. Yigong Shi. et al. Tsinghua University
ABSTRACT: Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat.
[more...]
Use: pLink



Special Enrichment Strategies Greatly Increase the Efficiency of Missing Proteins Identification from Regular Proteome Samples.
Journal of Proteome Research. 2015. Na Su. et al. Beijing Proteome Research Center
ABSTRACT: As part of the Chromosome-Centric Human Proteome Project (C-HPP) mission, laboratories all over the world have tried to map the entire missing proteins (MPs) since 2012. On the basis of the first and second Chinese Chromosome Proteome Database (CCPD 1.0 and 2.0) studies, we developed systematic enrichment strategies to identify MPs that fell into four classes: (1) low molecular weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), and (4) nucleic acid-associated proteins. Of 8845 proteins identified in 7 data sets, 79 proteins were classified as MPs.
[more...]
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Releasing N-glycan from Peptide N-terminus by N-terminal Succinylation Assisted Enzymatic Deglycosylation.
Scientific Reports. 2015. Yejing Weng. et al. Dalian Institute of Chemical Physics
ABSTRACT: Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests.
[more...]
Use: pBuild



Probing structures of large protein complexes using zero-length cross-linking.
Methods. 2015. Roland F. Rivera-Santiago. et al. The Wistar Institute
ABSTRACT: Structural mass spectrometry (MS) is a field with growing applicability for addressing complex biophysical questions regarding proteins and protein complexes. One of the major structural MS approaches involves the use of chemical cross-linking coupled with MS analysis (CX-MS) to identify proximal sites within macromolecules. Identified cross-linked sites can be used to probe novel protein–protein interactions or the derived distance constraints can be used to verify and refine molecular models.
[more...]
Use: pLink



Preparation of Hydrophilic monolithic capillary column by in situ photo-polymerization of N-vinyl-2-pyrrolidinone and acrylamide for highly selective and sensitive enrichment of N-linked glycopeptides.
Talanta. 2015. Hao Jiang. et al. Dalian Institute of Chemical Physics
ABSTRACT: In this study, a novel kind of amide functionalized hydrophilic monolith was synthesized by the in situ photo-polymerization of N-vinyl-2-pyrrolidinone (NVP), acrylamide (AM), and N, N’-methylenebisacrylamide (MBA) in a UV transparent capillary, and successfully applied for hydrophilic interaction chromatography (HILIC) based enrichment of N-linked glycopeptides. With 2 μg of the tryptic digests of IgG as the sample, after enrichment, 18 glycopeptides could be identified by MALDI-TOF/TOF MS analysis.
[more...]
Use: pBuild



Particulate capillary precolumns with double-end polymer monolithic frits for on-line peptide trapping and preconcentration.
Chinese Chemical Letters. 2015. Simin Xia. et al. Dalian Institute of Chemical Physics
ABSTRACT: In this work, a novel kind of particulate capillary precolumns with double-end polymer monolithic frits has been developed. Firstly, the polymer monolithic frit at one end was prepared via photo-initiated polymerization of a mixture of lauryl methacrylate and ethyleneglycol dimethacrylate with 1-propanol and 1,4-butanediol as porogens and 2,2-dimethoxy-2-phenylacetophenone as a photo-initiator in UV transparent coating capillary (100 μm i.d.). Subsequently, C18 particles (5 μm, 100 Å) were packed into the capillary, and sealed with the polymer monolithic frit at another end.
[more...]
Use: pBuild



Large-Scale Identification of Core-Fucosylated Glycopeptide Sites in Pancreatic Cancer Serum Using Mass Spectrometry.
Journal of Proteome Research. 2015. Zhijing Tan. et al. University of Michigan
ABSTRACT: Glycosylation has significant effects on protein function and cell metastasis, which are important in cancer progression. It is of great interest to identify site-specific glycosylation in search of potential cancer biomarkers. However, the abundance of glycopeptides is low compared to that of nonglycopeptides after trypsin digestion of serum samples, and the mass spectrometric signals of glycopeptides are often masked by coeluting nonglycopeptides due to low ionization efficiency.
[more...]
Use: pFind



Kojak: Efficient analysis of chemically cross-linked protein complexes.
Journal of Proteome Research. 2015. Michael R. Hoopmann. et al. Washington University
ABSTRACT: Protein chemical cross-linking and mass spectrometry enable the analysis of protein–protein interactions and protein topologies; however, complicated cross-linked peptide spectra require specialized algorithms to identify interacting sites. The Kojak cross-linking software application is a new, efficient approach to identify cross-linked peptides, enabling large-scale analysis of protein–protein interactions by chemical cross-linking techniques. The algorithm integrates spectral processing and scoring schemes adopted from traditional database search algorithms and can identify cross-linked peptides using many different chemical cross-linkers with or without heavy isotope labels.
[more...]
Use: pLink



Integrated SDS removal and protein digestion by hollow fiber membrane based device for SDS-assisted proteome analysis.
Talanta. 2015. Simin Xia. et al. Dalian Institute of Chemical Physics
ABSTRACT: In this work, a novel integrated sample preparation device for SDS-assisted proteome analysis was developed, by which proteins dissolved in 4% (w/v) SDS were first diluted by 50% methanol, and then SDS was online removed by a hollow fiber membrane interface (HFMI) with 50 mM ammonium bicarbonate (pH 8.0) as an exchange buffer, finally digested by an immobilized enzyme reactor (IMER). To evaluate the performance of such an integrated device, bovine serum albumin dissolved in 4% (w/v) SDS as a model sample was analyzed; it could be found that similar to that obtained by direct analysis of BSA digests without SDS (the sequence coverage of 60.3±1.0%, n=3), with HFMI as an interface for SDS removal, BSA was identified with the sequence coverage of 61.0±1.0% (n=3).
[more...]
Use: pXtract, pBuild



Identification of missing proteins defined by chromosome-centric proteome project in the cytoplasmic detergent-insoluble proteins.
Journal of Proteome Research. 2015. Yang Chen. et al. Jinan University
ABSTRACT: Finding protein evidence (PE) for protein coding genes is a primary task of the Phase I Chromosome-Centric Human Proteome Project (C-HPP). Currently, there are 2948 PE level 2–4 coding genes per neXtProt, which are deemed missing proteins in the human proteome. As most samples prepared and analyzed in the C-HPP framework were focusing on detergent soluble proteins, we posit that as a natural composition the cytoplasmic detergent-insoluble proteins (DIPs) represent a source of finding missing proteins.
[more...]
Use: pLabel



High-throughput screening of cellular redox sensors using modern redox proteomics approaches.
Proteomics. 2015. Jingwen Jiang. et al. Sichuan University
ABSTRACT: Cancer cells are characterized by higher levels of intracellular reactive oxygen species (ROS) due to metabolic aberrations. ROS are widely accepted as second messengers triggering pivotal signaling pathways involved in the process of cell metabolism, cell cycle, apoptosis, and autophagy. However, the underlying cellular mechanisms remain largely unknown. Recently, accumulating evidence has demonstrated that ROS initiate redox signaling through direct oxidative modification of the cysteines of key redox-sensitive proteins (termed redox sensors).
[more...]
Use: pLink



Finding Missing Proteins from the Epigenetically Manipulated Human Cell with Stringent Quality Criteria.
Journal of Proteome Research. 2015. Lijuan Yang. et al. Jinan University
ABSTRACT: The chromosome-centric human proteome project (C-HPP) has made great progress of finding protein evidence (PE) for missing proteins (PE2–4 proteins defined by the neXtProt), which now becomes an increasingly challenging field. As a majority of samples tested in this field were from adult tissues/cells, the developmental stage specific or relevant proteins could be missed due to biological source availability. We posit that epigenetic interventions may help to partially bypass such a limitation by stimulating the expression of the “silenced” genes in adult cells, leading to the increased chance of finding missing proteins. In this study, we established in vitro human cell models to modify the histone acetylation, demethylation, and methylation with near physiological conditions.
[more...]
Use: pLabel



Evaluation and Comparison of Aligners for De Novo Sequencing.
International Journal of Advanced Computer Technology. 2015. Simin Zhu. et al. Yunnan Minzu University
ABSTRACT: In high-throughput proteomics research of tandem mass spectrometry, de novo sequencing provides a novel method to interpret MS/MS data without any help of sequence database and discover new organisms. In this paper, we have systematically evaluated and compared the capability of mainstream de novo sequencing software via testing data sets which have been correctly identified by Mascot and Sequest, so we can intuitively find out the optimal de novo sequencing software for protein identification.
Use: pNovo



Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositolanchored High Density Lipoprotein-binding Protein 1.
THE JOURNAL OF BIOLOGICAL CHEMISTRY. 2015. Mart Reimund. et al. Tallinn University of Technology
ABSTRACT: GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P.
[more...]
Use: pLink



EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra.
Molecular & Cellular Proteomics. 2015. Zuo-Fei Yuan. et al. University of Pennsylvania
ABSTRACT: Histone post-translational modifications (PTMs) contribute to chromatin function through their chemical properties which influence chromatin structure, and their ability to recruit chromatin interacting proteins. Nanoflow liquid chromatography coupled with high resolution tandem mass spectrometry (nanoLC-MS/MS) has emerged as the most suitable technology for global histone modification analysis due to the high sensitivity and the high mass accuracy that provide confident identification.
[more...]
Use: pXtract



Drawbacks in the use of unconventional hydrophobic anhydrides for histone derivatization in bottom-up proteomics PTM analysis.
Proteomics. 2015. Simone Sidoli. et al. University of Pennsylvania
ABSTRACT: MS-based proteomics has become the most utilized tool to characterize histone PTMs. Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (<6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in RP LC, and such procedure also hampers the discovery of natural propionylation events.
[more...]
Use: pFind, pBuild



Direct energy transfer from themajor antenna to the photosystemII core complexes in the absence of minor antennae in liposomes.
Biochimica et Biophysica Acta. 2015. Ruixue Sun. et al. Institute of Botany, Chinese Academy of Sciences
ABSTRACT: Minor antennae of photosystem (PS) II, located between the PSII core complex and the major antenna (LHCII), are important components for the structural and functional integrity of PSII supercomplexes. In order to study the functional significance of minor antennae in the energetic coupling between LHCII and the PSII core, characteristics of PSII–LHCII proteoliposomes, with or without minor antennae, were investigated.
[more...]
Use: pLabel



CyanOmics: an integrated database of omics for the model cyanobacterium Synechococcus sp. PCC 7002.
the journal of biological databases and curation. 2015. Yaohua Yang. et al. Institute of Hydrobiology, Chinese Academy of Sciences
ABSTRACT: Cyanobacteria are an important group of organisms that carry out oxygenic photosynthesis and play vital roles in both the carbon and nitrogen cycles of the Earth. The annotated genome of Synechococcus sp. PCC 7002, as an ideal model cyanobacterium, is available. A series of transcriptomic and proteomic studies of Synechococcus sp. PCC 7002 cells grown under different conditions have been reported.
[more...]
Use: pFind



Convenient and precise strategy for mapping N-glycosylation sites using microwave-assisted acid hydrolysis and characteristic ions recognition.
Analytical Chemistry. 2015. Cheng Ma. et al. Georgia State University
ABSTRACT: N-glycosylation is one of the most prevalence protein post-translational modifications (PTM) which is involved in several biological processes. Alternation of N-glycosylation is associated with cellular malfunction and development of disease. Thus, investigation of protein N-glycosylation is crucial for diagnosis and treatment of disease. Currently, deglycosylation with peptide N-glycosidase F is the most commonly used technique in N-glycosylation analysis.
[more...]
Use: pFind, pBuild



Controlling Nonspecific Trypsin Cleavages in LC− MS/MS-Based Shotgun Proteomics Using Optimized Experimental Conditions.
Analyst. 2015. Pan Fang. et al. Fudan University
ABSTRACT: Trypsin has traditionally been used for enzymatic digestion during sample preparation in shotgun proteomics. The stringent specificity of trypsin is essential for accurate protein identification and quantification. But nonspecific trypsin cleavages are often observed in LC-MS/MS-based shotgun proteomics. To explore the extent of nonspecific trypsin cleavages, a series of biological systems including a standard protein mixture, Saccharomyces cerevisiae, human serum, human cancer cell lines and mouse brain were examined.
[more...]
Use: pFind



Conformational states of the full-length glucagon receptor.
Nature Communications. 2015. Linlin Yang. et al. Shanghai Institute of Materia Medica, Chinese Academy of Sciences
ABSTRACT: Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding.
[more...]
Use: pXtract, pLink



Chemical cross-linking and mass spectrometry to determine the subunit interaction network in a recombinant human SAGA HAT subcomplex.
Protein Science. 2015. Nha-Thi Nguyen-Huynh. et al. Université de Strasbourg
ABSTRACT: Understanding the way how proteins interact with each other to form transient or stable protein complexes is a key aspect in structural biology. In this study, we combined chemical cross-linking with mass spectrometry to determine the binding stoichiometry and map the protein-protein interaction network of a human SAGA HAT subcomplex. MALDI-MS equipped with high mass detection was used to follow the cross-linking reaction using bis[sulfosuccinimidyl] suberate (BS3) and confirm the heterotetrameric stoichiometry of the specific stabilized subcomplex. Cross-linking with isotopically labeled BS3 d0-d4 followed by trypsin digestion allowed the identification of intra- and intercross-linked peptides using two dedicated search engines: pLink and xQuest.
[more...]
Use: pLink



Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane.
PLOS One. 2015. lzabel Moraes. et al. Universidade de São Paulo
ABSTRACT: Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate.
[more...]
Use: pFind



A paired ions scoring algorithm based on Morpheus for simultaneous identification and quantification of proteome samples prepared by isobaric peptide termini labeling strategies.
PROTEOMICS. 2015. Shen Zhang. et al. Dalian Institute of Chemical Physics
ABSTRACT: The isobaric peptide termini labeling (IPTL) method is a promising strategy in quantitative proteomics for its high accuracy, while the increased complexity of MS2 spectra originated from the paired b, y ions has adverse effect on the identification and the coverage of quantification. Here, a paired ions scoring algorithm (PISA) based on Morpheus, a database searching algorithm specifically designed for high-resolution MS2 spectra, was proposed to address this issue.
[more...]
Use: pLabel



A Framework of De Novo Peptide Sequencing for Multiple Tandem Mass Spectra.
IEEE Transactions on nanobioscience. 2015. Yan Yan. et al. University of Saskatchewan Saskatoon
ABSTRACT: With tandem mass spectrometry (MS/MS), spectra can be generated by various fragmentation techniques including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD) and so on. At the same time, de novo sequencing using multiple spectra from the same peptide generated by different fragmentation techniques is becoming popular in proteomics studies.
[more...]
Use: pNovo




2014




蛋白质序列及其翻译后修饰鉴定的盲搜索算法评价.
科研信息化技术与应用. 2014. 黄潘育. et al. 中国科学院计算机网络信息中心超级计算中心
摘要:蛋白质翻译后修饰鉴定是计算蛋白质科学的重要研究方向,研发速度快和精度高的鉴定算法及软件,是该方向的一个主要问题。本文回顾了蛋白质翻译后修饰鉴定的各类算法,并主要对蛋白质序列及其翻译后修饰鉴定的盲搜索算法和软件进行研究。通过对具有代表性的两个盲搜索软件InsPecT和SIMS,以及限定性搜索软件pFind的对比分析,对盲搜索算法在蛋白质序列及其翻译后修饰方面的瓶颈、问题及未来发展趋势做出了评价。
Use: pFind



Visualization of arrestin recruitment by a G-protein-coupled receptor.
Nature. 2014. Arun K. Shukla. et al. Stanford University School of Medicine
ABSTRACT: G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)–G-protein complex, has provided novel insights into the structural basis of receptor activation.
[more...]
Use: pLink



Various Conotoxin Diversifications Revealed by a Venomic Study of Conus flavidus.
Molecular & Cellular Proteomics. 2014. Aiping Lu. et al. Tongji University
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.
[more...]
Use: pXtract, pFind, pBuild, pLabel



UvrD facilitates DNA repair by pulling RNA polymerase backwards.
Nature. 2014. Vitaly Epshtein. et al. New York University School of Medicine
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.
[more...]
Use: pLink, pLabel



Transferred subgroup false discovery rate for rare post-translational modifications detected by mass spectrometry.
Molecular & Cellular Proteomics. 2014. Yan Fu. et al. Beijing Proteome Research Center
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.
[more...]
Use: pFind



Structural Characterization by Cross-linking Reveals the Detailed Architecture of a Coatomer-related Heptameric Module from the Nuclear Pore Complex.
Molecular & Cellular Proteomics. 2014. Yi Shi. et al. The Rockefeller University
ABSTRACT: Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for the structural characterization of such complexes that has at its center chemical cross-linking with mass spectrometric readout.
[more...]
Use: pXtract, pLink



Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays.
Journal of Proteomics. 2014. Ana O. Tiroli-Cepeda. et al. University of Campinas
ABSTRACT: Hsp70 cycles from an ATP-bound state, in which the affinity for unfolded polypeptides is low, to an ADP-bound state, in which the affinity for unfolded polypeptides is high, to assist with cell proteostasis. Such cycling also depends on co-chaperones because these proteins control both the Hsp70 ATPase activity and the delivery of unfolded polypeptide chains. Although it is very important, structural information on the entire protein is still scarce.
[more...]
Use: pLink



Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry.
Analytical Chemistry. 2014. Qichen Cao. et al. Beijing Institute of Radiation Medicine
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 α-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 MS(3) scanning under collision-induced dissociation (CID) by ion trap mass spectrometry.
[more...]
Use: pFind



Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.
Biochimica et Biophysica Acta (BBA). 2014. Long Zhao. et al. Beijing Institute of Radiation Medicine
ABSTRACT: Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo.
[more...]
Use: pXtract



Reconstitution of active human core Mediator complex reveals a critical role of the MED14 subunit.
nature structural & molecular biology. 2014. Murat A Cevher. et al. Rockefeller University
ABSTRACT: The evolutionarily conserved Mediator complex is a critical coactivator for RNA polymerase II (Pol II)-mediated transcription. Here we report the reconstitution of a functional 15-subunit human core Mediator complex and its characterization by functional assays and chemical cross-linking coupled to MS (CX-MS). Whereas the reconstituted head and middle modules can stably associate, basal and coactivator functions are acquired only after incorporation of ​MED14 into the bimodular complex.
[more...]
Use: pLink



Proteome Informatics Research Group(iPRG)_2012: A Study on Detecting Modified Peptides in a Complex Mixture.
Biochemistry and Molecular Biology. 2014. Chalkley, RJ. et al. University of California
ABSTRACT: The proteome informatics research group of the Association of Biomolecular Resource Facilities conducted a study to assess the community's ability to detect and characterize peptides bearing a range of biologically occurring post-translational modifications when present in a complex peptide background. A data set derived from a mixture of synthetic peptides with biologically occurring modifications combined with a yeast whole cell lysate as background was distributed to a large group of researchers and their results were collectively analyzed.
[more...]
Use: pFind



Proteogenomic analysis and global discovery of posttranslational modifications in prokaryotes.
PNAS. 2014. Ming-kun Yang. et al. Institute of Hydrobiology, Chinese Academy of Sciences
ABSTRACT: We describe an integrated workflow for proteogenomic analysis and global profiling of posttranslational modifications (PTMs) in prokaryotes and use the model cyanobacterium Synechococcus sp. PCC 7002 (hereafter Synechococcus 7002) as a test case. We found more than 20 different kinds of PTMs, and a holistic view of PTM events in this organism grown under different conditions was obtained without specific enrichment strategies. Among 3,186 predicted protein-coding genes, 2,938 gene products (>92%) were identified.
[more...]
Use: pFind



Preparation of high efficiency and low carry-over immobilized enzymatic reactor with methacrylic acid–silica hybrid monolith asmatrix for on-line protein digestion.
Journal of Chromatography A. 2014. Huiming Yuan. et al. Dalian Institute of Chemical Physics
ABSTRACT: In this work, a novel kind of organic-silica hybrid monolith based immobilized enzymatic reactor (IMER) was developed. The monolithic support was prepared by a single step "one-pot" strategy via the polycondensation of tetramethoxysilane and vinyltrimethoxysilane and in situ copolymerization of methacrylic acid and vinyl group on the precondensed siloxanes with ammonium persulfate as the thermal initiator. Subsequently, the monolith was activated by N-(3-dimethylaminopropyl) - N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), followed by the modification of branched polyethylenimine (PEI) to improve the hydrophilicity.
[more...]
Use: pBuild



Phosphoproteomic Analysis Provides Novel Insights into Stress Responses in Phaeodactylum tricornutum, a Model Diatom.
Journal of Proteome Research. 2014. Zhuo Chen. et al. Institute of Hydrobiology, Chinese Academy of Sciences
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.
[more...]
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Peptidomics combined with cDNA library unravel the diversity of centipede venom.
JOURNAL OF PROTEOMICS. 2014. Mingqiang Rong. et al. BGI-Shenzhen
ABSTRACT: Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins were more complicated than any other animal toxins and even exhibited large differences in homologues.
[more...]
Use: pLabel



NovoHCD: De novo Peptide Sequencing From HCD Spectra.
IEEE Transactions on nanobioscience. 2014. Yan Yan. et al. University of Saskatchewan Saskatoon
ABSTRACT: In recent years, de novo peptide sequencing from mass spectrometry data has developed as one of the major peptide identification methods with the emergence of new instruments and advanced computational methods. However, there are still limitations to this method; for example, the typically used spectrum graph model cannot represent all the information and relationships inherent in tandem mass spectra (MS/MS spectra). Here, we present a new method named NovoHCD which applies a spectrum graph model with multiple types of edges (called a multi-edge graph), and integrates into it amino acid combination (AAC) information and peptide tags.
[more...]
Use: pNovo



NovoExD: De novo peptide sequencing for ETD/ECD spectra.
IEEE Transactions on nanobioscience. 2014. Yan Yan. et al. University of Saskatchewan Saskatoon
ABSTRACT: De novo peptide sequencing using tandem mass spectrometry (MS/MS) data has become a major computational method for sequence identification in recent years. With the development of new instruments and technology, novel computational methods have emerged with enhanced performance. However, there are only a few methods focusing on ECD/ETD spectra, which mainly contain variants of c-ions and z-ions.
[more...]
Use: pNovo



N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid Chromatography-Based Enrichment, Parallel Deglycosylation, and Mass Spectrometry.
Journal of Proteome Research. 2014. Liwei Cao. et al. Dalian Institute of Chemical Physics
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.
[more...]
Use: pXtract, pBuild



Molecular architecture and function of the SEA complex, a modulator of the TORC1 pathway.
Molecular & Cellular Proteomics . 2014. Romain Algret. et al. Universite´ Paris-Sud 11
ABSTRACT: The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy, and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular levels of SEA complex members.
[more...]
Use: pLink



Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface.
THE JOURNAL OF BIOLOGICAL CHEMISTRY. 2014. Wilson Wong. et al. Imperial College of Science, Technology and Medicine
ABSTRACT: Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament.
[more...]
Use: pLink



Matching Cross-linked Peptide Spectra: Only as Good as the Worse Identification.
Molecular & Cellular Proteomics. 2014. Michael J. Trnka. et al. University of California San Francisco
ABSTRACT: Chemical cross-linking mass spectrometry identifies interacting surfaces within a protein assembly through labeling with bifunctional reagents and identifying the covalently modified peptides. These yield distance constraints that provide a powerful means to model the three-dimensional structure of the assembly. Bioinformatic analysis of cross-linked data resulting from large protein assemblies is challenging because each cross-linked product contains two covalently linked peptides, each of which must be correctly identified from a complex matrix of potential confounders.
[more...]
Use: pLink



In Vivo Proximity Labeling for the Detection of Protein–Protein and Protein–RNA Interactions.
Journal of Proteome Research. 2014. David B Beck. et al. Department of Cell and Developmental Biology, Epigenetics Program
ABSTRACT: Accurate and sensitive detection of protein–protein and protein–RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo.
[more...]
Use: pParse, pFind



Identification of Human Tissue Kallikrein 6 as a Potential Marker of Laryngeal Cancer Based on the Relevant Secretory/Releasing Protein Database.
Disease Markers. 2014. Ying Zhang. et al. Chinese Academy of Medical Sciences
ABSTRACT: This study was aimed to create a large-scale laryngeal cancer relevant secretory/releasing protein database and further discover candidate biomarkers. Primary tissue cultures were established using tumor tissues and matched normal mucosal tissues collected from four laryngeal cancer patients. Serum-free conditioned medium (CM) samples were collected. These samples were then sequentially processed by SDS-PAGE separation, trypsin digestion, and LC-MS/MS analysis.
[more...]
Use: pFind



Hunting for Unexpected Post-Translational Modifications by Spectral Library Searching with Tier-Wise Scoring.
Journal of Proteome Research. 2014. Chun Wai Manson Ma. et al. Hong Kong University of Science and Technology
ABSTRACT: Discovering novel post-translational modifications (PTMs) to proteins and detecting specific modification sites on proteins is one of the last frontiers of proteomics. At present, hunting for post-translational modifications remains challenging in widely practiced shotgun proteomics workflows due to the typically low abundance of modified peptides and the greatly inflated search space as more potential mass shifts are considered by the search engines.
[more...]
Use: pMatch



Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications.
Journal of Proteome Research. 2014. Zuo-Fei Yuan. et al. University of Pennsylvania
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.
[more...]
Use: pParse, pFind



Enhanced Identification of Zero-Length Chemical Cross-Links Using Label-Free Quantitation and High-Resolution Fragment Ion Spectra.
Journal of Proteome Research. 2014. Sira Sriswasdi. et al. Wistar Institute
ABSTRACT: Chemical cross-linking coupled to mass spectrometry provides structural information that is useful for probing protein conformations and providing experimental support for molecular models. “Zero-length” cross-links have greater value for these applications than longer cross-links because they provide more stringent distance constraints. However, this method is less commonly utilized because it cannot take advantage of isotopic labels, MS-labile bonds, or enrichment tags to facilitate identification.
[more...]
Use: pLink



Dimerization of isolated Pseudomonas aeruginosa lipopolysaccharide transporter component LptA.
Biochmical and Biophysical Research Communications. 2014. Adam B. Shapiro. et al. Infection Innovative Medicines Unit/Reagents
ABSTRACT: LptA is a soluble periplasmic component of the lipopolysaccharide (LPS) transport system of Gram-negative bacteria that transports newly synthesized LPS from the inner membrane to the outer leaflet of the outer membrane. LptA links the inner membrane components (LptBFGC) to the outer membrane components (LptDE), but it is uncertain whether LptA is a freely moving LPS shuttle or part of a stable trans-periplasm structure. Escherichiacoli LptA forms highly polymerized head-to-tail oligomers in solution, but dimers in vivo.
[more...]
Use: pLink, pLabel



Determining Protein Subcellular Localization in Mammalian Cell Culture with Biochemical Fractionation and iTRAQ 8-Plex Quantification.
Shotgun Proteomics: Methods and Protocols, Methods in Molecular Biology. 2014. Andy Christoforou. et al. University of Cambridge
ABSTRACT: Protein subcellular localization is a fundamental feature of posttranslational functional regulation. Traditional microscopy based approaches to study protein localization are typically of limited throughput, and dependent on the availability of antibodies with high specificity and sensitivity, or fluorescent fusion proteins. In this chapter we describe how Localization of Organelle Proteins by Isotope Tagging (LOPIT), a mass spectrometry based workflow coupling biochemical fractionation and iTRAQ™ 8-plex quantification, can be applied for the high-throughput characterization of protein localization in a mammalian cell culture line.
Use: pXtract



Dendrimer-grafted graphene oxide nanosheets as novel support for trypsin immobilization to achieve fast on-plate digestion of proteins.
Talanta. 2014. Bo Jiang. et al. Dalian Institute of Chemical Physics
ABSTRACT: In this study, dendrimer grafted graphene oxide nanosheets (dGO) were prepared by covalent reaction. The successful synthesis of dGO was confirmed by Fourier-transform infrared spectra, Raman spectra, Thermo gravimetric analysis and Zeta potential. Taking advantages of large surface area, excellent biocompatibility and abundant functional groups, dGO provided an ideal substrate for trypsin immobilization. Trypsin-linked dGO was synthesized through covalent bonding using glutaraldehyde as coupling agents.
[more...]
Use: pBuild



De novo identification and quantification of single amino-acid variants in human brain.
Journal of Molecular Cell Biology. 2014. Zhi-Duan Su. et al. Shanghai Institutes for Biological Sciences
ABSTRACT: The detection of single amino-acid variants (SAVs) usually depends on single-nucleotide polymorphisms (SNPs) database. Here, we describe a novel method that discovers SAVs at proteome level independent of SNPs data. Using mass spectrometry-based de novo sequencing algorithm, peptide-candidates are identified and compared with theoretical protein database to generate SAVs under pairing strategy, which is followed by database re-searching to control false discovery rate.
[more...]
Use: pNovo



Complex Proteomes Analysis Using Label-Free Mass Spectrometry-Based Quantitative Approach Coupled with Biomedical Knowledge.
International Federation for Information Processing. 2014. Chao Pan. et al. Zhejiang University
ABSTRACT: Label-free quantitative proteomics based on mass spectrometry plays an essential role in large-scale analysis of complex proteomes. Meanwhile, quantitative proteomics is not only a way for data processing, but also an important approach for exploring protein functions and interactions in a large-scale manner. An effective method combining quantitation and qualification should be built.
[more...]
Use: pFind



Combinatorial Approach for Large-scale Identification of Linked Peptides from Tandem Mass Spectrometry Spectra.
Molecular & Cellular Proteomics. 2014. Jian Wang. et al. University of California
ABSTRACT: The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein–protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical.
[more...]
Use: pLink



Architecture of the Saccharomyces cerevisiae SAGA transcription coactivator complex.
The EMBO Journal. 2014. Yan Han. et al. Institute for Systems Biology, Seattle
ABSTRACT: The conserved transcription coactivator SAGA is comprised of several modules that are involved in activator binding, TBP binding, histone acetylation (HAT) and deubiquitination (DUB). Crosslinking and mass spectrometry, together with genetic and biochemical analyses, were used to determine the molecular architecture of the SAGA‐TBP complex. We find that the SAGA Taf and Taf‐like subunits form a TFIID‐like core complex at the center of SAGA that makes extensive interactions with all other SAGA modules.
[more...]
Use: pLink



Architecture of the Saccharomyces cerevisiae RNA polymerase I Core Factor complex.
Nature Structural & Molecular Biology. 2014. Bruce A Knutso. et al. Fred Hutchinson Cancer Research Center, Seattle
ABSTRACT: Core Factor (CF) is a conserved RNA polymerase (Pol) I general transcription factor comprising ​Rrn6, ​Rrn11 and the ​TFIIB-related subunit ​Rrn7. CF binds ​TATA-binding protein (​TBP), Pol I and the regulatory factors ​Rrn3 and upstream activation factor. We used chemical cross-linking–MS to determine the molecular architecture of CF and its interactions with ​TBP. The CF subunits assemble through an interconnected network of interactions between five structural domains that are conserved in orthologous subunits of the human PolI factor SL1.
[more...]
Use: pLink



Acetylome Analysis Reveals Diverse Functions of Lysine Acetylation in Mycobacterium tuberculosis.
Molecular & Cellular Proteomics. 2014. Fengying Liu. et al. Institute of Hydrobiology, Chinese Academy of Sciences
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.
[more...]
Use: pFind



Accelerating the scoring module of mass spectrometry-based peptide identification using GPUs.
BMC Bioinformatics. 2014. You Li. et al. 香港浸会大学
ABSTRACT: Tandem mass spectrometry-based database searching is currently the main method for protein identification in shotgun proteomics. The explosive growth of protein and peptide databases, which is a result of genome translations, enzymatic digestions, and post-translational modifications (PTMs), is making computational efficiency in database searching a serious challenge.
[more...]
Use: pFind



Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry.
Analytical Chemistry. 2014. Qichen Cao. et al. Beijing Institute of Radiation Medicine
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 α-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value.
[more...]
Use: pFind



A precise approach in large scale core-fucosylated glycoprotein identification with low-and high-normalized collision energy.
Journal of Proteomics. 2014. Cheng Ma. et al. Georgia State University
ABSTRACT: The core fucosylation (CF) of N-glycoproteins plays important roles in regulating protein functions during biological development, and it has also been shown to be up-regulated in several high metastasis cancer cell lines. Therefore, global profiling and quantitative characterization of CF-glycoproteins may reveal potent biomarkers for clinical applications.
[more...]
Use: pFind, pBuild



1-Dodecyl-3-Methylimidazolium Chloride-Assisted Sample Preparation Method for Efficient Integral Membrane Proteome Analysis.
Analytical Chemistry. 2014. Qun Zhao. et al. Dalian Institute of Chemical Physics
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.
[more...]
Use: pXtract, pBuild




2013




原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用.
色谱. 2013. 周廉淇. et al. 北京蛋白质组研究中心
摘要:针对传统溶液酶解存在的酶解时间较长、酶自切物干扰以及蛋白酶不能重复使用等缺陷,我们通过电子转移生成催化剂的原子转移自由基聚合法修饰银丝,并以其做为载体制备了一种新型的固定化酶反应器。采用标准蛋白质和质谱考察了银丝固定化酶反应器的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的银丝固定化酶反应器的酶解效率较高,酶解标准蛋白BSA 20 min后,肽段的氨基酸序列覆盖率可达93%,好于传统溶液酶切方法酶切16 h得到的79%的覆盖率;使用该固定化酶反应器在一个月内8次酶解BSA所得的氨基酸序列覆盖率在89%到97%之间,平均覆盖率为94%,显示出其具有良好的稳定性。
[more...]
Use: pFind, pBuild



UniNovo: a universal tool for de novo peptide sequencing.
Bioinformatics. 2013. Kyowon Jeong. et al. University of California
ABSTRACT: Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but de novo peptide sequencing algorithms to analyze tandem mass (MS/MS) spectra are lagging behind. Although existing de novo sequencing tools perform well on certain types of spectra [e.g. Collision Induced Dissociation (CID) spectra of tryptic peptides], their performance often deteriorates on other types of spectra, such as Electron Transfer Dissociation (ETD), Higher-energy Collisional Dissociation (HCD) spectra or spectra of non-tryptic digests.
[more...]
Use: pNovo



Thioredoxin and Thioredoxin Reductase Control Tissue Factor Activity by Thiol Redox-dependent Mechanism.
THE JOURNAL OF BIOLOGICAL CHEMISTRY. 2013. Pei Wang. et al. University of Chinese Academy of Sciences
ABSTRACT: Abnormally enhanced tissue factor (TF) activity is related to increased thrombosis risk in which oxidative stress plays a critical role. Human cytosolic thioredoxin (hTrx1) and thioredoxin reductase (TrxR), also secreted into circulation, have the power to protect against oxidative stress. However, the relationship between hTrx1/TrxR and TF remains unknown. Here we show reversible association of hTrx1 with TF in human serum and plasma samples.
[more...]
Use: pFind



Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma.
Science China Life Sciences, Springer. 2013. Yang Lei. et al. 中国医学科学院
ABSTRACT: For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins.
[more...]
Use: pFind



N-linked glycoproteome profiling of human serum using tandem enrichment and multiple fraction concatenation.
Electrophoresis. 2013. Cheng Ma. et al. Beijing Institute of Radiation Medicine
ABSTRACT: N-linked glycosylation is an important protein posttranslational modification that is involved in numerous biological processes. Different methods, including chemical reaction and affinity interaction, have been developed to enrich glycosylated peptides or proteins from biological systems. However, due to the common occurrence of low glycosites occupancy in proteins and the low efficiency of enrichment approaches, only a small fraction of protein glycosites have been reported.
[more...]
Use: pFind



Method for Rapid Protein Identification in a Large Database.
BioMed Research International. 2013. Wenli Zhang. et al. Institute of Computing Technology, Chinese Academy of Sciences
ABSTRACT: Protein identification is an integral part of proteomics research. The available tools to identify proteins in tandem mass spectrometry experiments are not optimized to face current challenges in terms of identification scale and speed owing to the exponential growth of the protein database and the accelerated generation of mass spectrometry data, as well as the demand for nonspecific digestion and post-modifications in complex-sample identification.
[more...]
Use: pFind



Mass Defect-Based Pseudo-Isobaric Dimethyl Labeling for Proteome Quantification.
Analytical Chemistry. 2013. Yuan Zhou. et al. Dalian Institute of Chemical Physics
ABSTRACT: Discovering differentially expressed proteins in various biological samples requires proteome quantification methods with accuracy, precision, and wide dynamic range. This study describes a mass defect-based pseudo-isobaric dimethyl labeling (pIDL) method based on the subtle mass defect differences between 12C/13C and 1H/2H. Lys-C protein digests were labeled with CD2O/13CD2O and reduced with NaCNBD3/NaCNBH3 as heavy and light isotopologues, respectively.
[more...]
Use: pXtract



Identification of b-/y-ions in MS/MS spectra using a two stage neural network.
Proteome science. 2013. James P Cleveland. et al. University of South Carolina
ABSTRACT: Independent of the approach used, the ability to correctly interpret tandem MS data depends on the quality of the original spectra. Even in the case of the highest quality spectra, the majority of spectral peaks can not be reliably interpreted. The accuracy of sequencing algorithms can be improved by filtering out such 'noise' peaks. Preprocessing MS/MS spectra to select informative ion peaks increases accuracy and reduces the processing time.
[more...]
Use: pNovo



Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris.
Biotechnology Letters. 2013. Da Lei. et al. Nanchang University
ABSTRACT: Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff’s base staining and Endo H digestion. Moreover, the deglycosylated protein’s molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI–TOF–MS, and the hyperglycosylation extent was 21 %.
[more...]
Use: pFind



Global Phosphoproteomic Analysis Reveals Diverse Functions of Serine/Threonine/Tyrosine Phosphorylation in the Model Cyanobacterium Synechococcus sp. Strain PCC 7002.
Journal of proteome. 2013. Ming-kun Yang. et al. Institute of Hydrobiology, Chinese Academy of Sciences
ABSTRACT: Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser, Thr, and Tyr residues.
[more...]
Use: pFind, pBuild, pLabel



Fragmentation Pattern of Glycated Peptides Derived from D-glucose by Tandem Mass Spectrometry.
2013 ICME INTERNATIONAL CONFERENCE ON COMPLEX MEDICAL ENGINEERING (CME). 2013. Rui Su. et al. Beijing Institute of Technology
ABSTRACT: Evidence shows that non-enzymatic glycation plays an important role in the development of diabetes, diabetes-related complications and neurodegenerative diseases. Mass spectrometric methods have been used for non-enzymatic glycation of proteins. At present, CID remains the major fragmentation method for peptide sequencing. In this study, we utilized synthesized peptide models to study fragmentation spectra of glycated peptides, which can be easily studied using ESI-MS.
[more...]
Use: pFind, pBuild, pLabel



Cross-linking and mass spectrometry methodologies to facilitate structural biology finding a path through the maze.
Journal of Structural and Functional Genomics. 2013. Eric D. Merkley. et al. Biological Sciences Division, Pacific Northwest
ABSTRACT: Multiprotein complexes, rather than individual proteins, make up a large part of the biological macromolecular machinery of a cell. Understanding the structure and organization of these complexes is critical to understanding cellular function. Chemical cross-linking coupled with mass spectrometry is emerging as a complementary technique to traditional structural biology methods and can provide low-resolution structural information for a multitude of purposes, such as distance constraints in computational modeling of protein complexes.
[more...]
Use: pLink



Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment.
Anal Bioanal Chem. 2013. Xinyuan Zhao. et al. Beijing Institute of Radiation Medicine
ABSTRACT: Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions.
[more...]
Use: pFind



Biphasic Microreactor for Efficient Membrane Protein Pretreatment with a Combination of Formic Acid Assisted Solubilization, On-Column pH Adjustment, Reduction, Alkylation, and Tryptic Digestion.
Analytical Chemistry. 2013. Qun Zhao. et al. Dalian Institute of Chemical Physics
ABSTRACT: Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an “SCX-SAX” biphasic capillary column.
[more...]
Use: pXtract, pBuild



A multi-edge graph based de novo peptide sequencing method for HCD spectra.
IEEE International Conference on Bioinformatics and Biomedicine. 2013 . Yan Yan. et al. University of Saskatchewan Saskatoon
ABSTRACT: In recent years, de novo peptide sequencing from mass spectrometry data has developed as one of the major peptide identification methods with the emergence of new instruments and advanced computational methods. However, there are still limitations to this method; for example, the typically used spectrum graph model cannot represent all the information and relationships inherent in tandem mass spectra (MS/MS spectra). Here, we present a new spectrum graph model with multiple types of edges (called a multi-edge graph), and integrate amino acid combination (AAC) information and peptide tags into it for peptide sequencing.
[more...]
Use: pNovo




2012




基于固相萃取的微量糖蛋白糖基化修饰表征技术的建立.
分析化学. 2012. 江静. et al. 军事医学科学院放射与辐射医学研究所
摘要: 结合自制亲水固相萃取富集柱和生物质谱鉴定技术,实现了糖基化蛋白质核糖核酸酶B的糖含量测定、糖基化位点确认、聚糖富集及结构表征,以及不同糖型相对丰度分析。结果表明: 其糖含量8.47%,糖基化位点为34位的Asn,糖链主要为5种高甘露糖型结构(Man5-9GlcNAc2)。所建立的HILIC富集技术,有利于针对微量生物样本,如生物工程药物糖蛋白及重要功能糖蛋白,开展位点特异性糖链结构解析,为糖蛋白质的药效或功能研究提供线索。
Use: pLabel



The ovarian cancer-derived secretory/releasing proteome: A repertoire of tumor markers.
Proteomics. 2012. Ying Zhang. et al. Chinese Academy of Medical Sciences
ABSTRACT: Ovarian cancer is the most lethal gynecological malignancy worldwide, and early detection of this disease using serum or plasma biomarkers may improve its clinical outcome. In the present study, a large scale protein database derived from ovarian cancer was created to enable tumor marker discovery.
[more...]
Use: pFind



Speeding up Scoring Module of Mass Spectrometry Based Protein Identification by GPU.
High Performance Computing and Communication & 2012 IEEE 9th International Conference on Embedded Software and Systems (HPCC-ICESS). 2012. You Li. et al. Hong Kong Baptist University Kowloon Tong
ABSTRACT: Database searching is a main method for protein identification in shotgun proteomics, and many research efforts are dedicated to improving its effectiveness. However, the efficiency of database searching is facing a serious challenge, due to the ever fast growth of protein and peptide databases resulted from genome translations, enzymatic digestions, and post-translational modifications (PTMs).
[more...]
Use: pFind



NSI and NSMT: usages of MS/MS fragment ion intensity for sensitive differential proteome detection and accurate protein fold change calculation in relative label-free proteome quantification.
Analyst. 2012. Qi Wu. et al. Dalian Institute of Chemical Physics
ABSTRACT: Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies.
[more...]
Use: pXtract, pBuild



Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin).
PNAS. 2012. Yanmei Zhao. et al. Institute of Biophysics, Chinese Academy of Sciences
ABSTRACT: Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions.
[more...]
Use: pNovo



Integrated Platform for Proteome Profiling with Combination of Microreversed Phase Based Protein and Peptide Separation via Online Solvent Exchange and Protein Digestion.
Analytical chemistry. 2012. Huiming Yuan. et al. Dalian Institute of Chemical Physics
ABSTRACT: An online integrated platform for proteome profiling was established, with the combination of protein separation by microreversed phase liquid chromatography (μRPLC), online acetonitrile (ACN) removal, and pH adjustment by a hollow fiber membrane interface (HFMI), online digestion by an immobilized enzymatic microreactor (IMER), as well as peptide separation and proteins identification by μRPLC or nano-RPLC-electrospray ionization tandem mass spectrometry (μRPLC-ESI-MS/MS).
[more...]
Use: pXtract, pBuild



Improved proteomic analysis pipeline for LC-ETD-MS/MS using charge enhancing methods.
Molecular BioSystems. 2012. Liqi Xie. et al. Fudan University
ABSTRACT: Electron transfer dissociation (ETD) is a useful and complementary activation method for peptide fragmentation in mass spectrometry. However, ETD spectra typically receive a relatively low score in the identifications of 2+ ions. To overcome this challenge, we, for the first time, systematically interrogated the benefits of combining ion charge enhancing methods (dimethylation, guanidination, m-nitrobenzyl alcohol (m-NBA) or Lys-C digestion) and differential search algorithms (Mascot, Sequest, OMSSA, pFind and X!Tandem).
[more...]
Use: pFind



Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion.
Journal of Chromatography A. 2012. Bo Jiang. et al. Dalian Institute of Chemical Physics
ABSTRACT: In this paper, magnetic Fe3O4 nanoparticles modified graphene oxide nanocomposites (GO–CO–NH–Fe3O4) were prepared by covalent bonding, via the reaction between the amino groups of fuctionalized Fe3O4 and the carboxylic groups of GO, confirmed by Fourier-transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. With GO–CO–NH–Fe3O4 as a novel substrate, trypsin was immobilized via π–π stacking and hydrogen bonding interaction, and the binding capacity of trypsin reached as high as 0.275 mg/mg.
[more...]
Use: pBuild



Determination of monoisotopic masses of chimera spectra from high-resolution mass spectrometric data by use of isotopic peak intensity ratio modeling.
Rapid Communications in Mass Spectrometry. 2012. Ming Niu. et al. Beijing Institute of Radiation Medicine
ABSTRACT: Chimera spectra make it challenging to identify proteins in complex mixtures by LC/MS/MS. Approximately half of the spectra collected are chimera spectra even when high-resolution tandem mass spectrometry is used. Chimera spectra are generated from the co-fragmentation of different co-elute peptides, and it is often difficult to distinguish monoisotopic precursors of these peptides from each other.In this paper, we propose a peak intensity ratio-based monoisotopic peak determination algorithm (PIRMD) to distinguish different monoisotopic precursors of chimera spectra.
[more...]
Use: pParse, pBuild, pLabel



A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue.
Journal of Chromatography A. 2012. Hao Jiang .et al. Dalian Institute of Chemical Physics
ABSTRACT: In this work, a novel kind of N-vinyl-2-pyrrolidinone (NVP) modified poly acrylic ester microspheres was prepared, followed by trypsin immobilization to prepare a hydrophilic immobilized enzyme reactor (IMER), to achieve highly efficient protein digestion with low peptide residue. The nonspecific adsorption of peptides on such an IMER was evaluated by the in sequence digestion of bovine serum albumin (BSA) and myoglobin. Without NVP modification, both proteins could be identified after digestion by a 5 cm-length IMER, but 18 peptides of BSA were found in the digests of myoglobin caused by the nonspecific adsorption of the matrix.
[more...]
Use: pXtract, pBuild




2011




Inverse Regulation in the Metabolic Genes pckA and metE Revealed by Proteomic Analysis of the Salmonella RcsCDB Regulon.
Journal of Proteome Research. 2011. Alberto Paradela. et al. Departamento de Biotecnología Microbiana
ABSTRACT: The RcsC, RcsD, and RcsB proteins compose a system used by enteric bacteria to sense envelope stress. Signal transmission occurs from the sensor RcsC to the transcriptional regulator RcsB. Accessory proteins, such as IgaA, are known to adjust the response level. In a previous transcriptomic study, we uncovered 85 genes differentially expressed in Salmonella enterica serovar Typhimurium igaA mutants. Here, we extended these observations to proteomics by performing differential isotope-coded protein labeling (ICPL) followed by liquid chromatography-electrospray ionization tandem mass spectrometry. Five-hundred five proteins were identified and quantified, with 75 of them displaying significant changes in response to alterations in the RcsCDB system.
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Use: pScan



Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction.
Skeletal Muscle. 2011. Suzanne E Mate. et al. George Washington University
ABSTRACT: During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins.Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction.
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Use: pXtract



Fragmentation and Site-Specific Quantification of Core Fucosylated Glycoprotein by Multiple Reaction Monitoring-Mass Spectrometry.
Analytical chemistry. 2011. Yan Zhao. et al. Beijing Institute of Radiation Medicine
ABSTRACT: Glycosylation modifications of proteins have been attracting increasing attention due to their roles in the physiological and pathological processes of the cell. Core fucosylation (CF), one special type of glycan structure in glycoproteins, has been linked with tumorigenesis. The study of protein glycosylation has been hindered by the technical challenges caused by the microheterogeneity of glycan modifications. In commonly used methods, sugar chains on the peptide were released using endoglycosidase, and the glycan and peptides were analyzed separately with mass spectrometry. Although mass spectrometric analysis can be performed easily in this way, an increase in false positives when assigning glycosites was inevitable.
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Use: pLabel



A Systematic Analysis of Eluted Fraction of Plasma Post Immunoaffinity Depletion: Implications in Biomarker Discovery.
PLOS ONE. 2011. Amit Kumar Yadav. et al. Institute of Genomics and Integrative Biology
ABSTRACT: Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins.
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Use: pLabel