pFind Studio: a computational solution for mass spectrometry-based proteomics
2018
BioMed Research International2018. An, Zhiwu et al.
State Key Laboratory of Bioactive Molecules and Druggability Assessment, International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development (MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West, 510632 Guangzhou, China
ABSTRACT:Confident characterization of intact glycopeptides is a challenging task in mass spectrometry-based glycoproteomics due to microheterogeneity of glycosylation, complexity of glycans, and insufficient fragmentation of peptide bones. Open mass spectral library search is a promising computational approach to peptide identification, but its potential in the identification of glycopeptides has not been fully explored. Here we present pMatchGlyco, a new spectral library search tool for intact N-linked glycopeptide identification using high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) data. In pMatchGlyco, (1) MS/MS spectra of deglycopeptides are used to create spectral library, (2) MS/MS spectra of glycopeptides are matched to the spectra in library in an open (precursor tolerant) manner and the glycans are inferred, and (3) a false discovery rate is estimated for top-scored matches above a threshold. The efficiency and reliability of pMatchGlyco were demonstrated on a data set of mixture sample of six standard glycoproteins and a complex glycoprotein data set generated from human cancer cell line OVCAR3.
Use: pFind; pParse; pGlyco
BioMed Research International2018. An, Zhiwu et al.
State Key Laboratory of Bioactive Molecules and Druggability Assessment, International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development (MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West, 510632 Guangzhou, China
ABSTRACT:Confident characterization of intact glycopeptides is a challenging task in mass spectrometry-based glycoproteomics due to microheterogeneity of glycosylation, complexity of glycans, and insufficient fragmentation of peptide bones. Open mass spectral library search is a promising computational approach to peptide identification, but its potential in the identification of glycopeptides has not been fully explored. Here we present pMatchGlyco, a new spectral library search tool for intact N-linked glycopeptide identification using high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS) data. In pMatchGlyco, (1) MS/MS spectra of deglycopeptides are used to create spectral library, (2) MS/MS spectra of glycopeptides are matched to the spectra in library in an open (precursor tolerant) manner and the glycans are inferred, and (3) a false discovery rate is estimated for top-scored matches above a threshold. The efficiency and reliability of pMatchGlyco were demonstrated on a data set of mixture sample of six standard glycoproteins and a complex glycoprotein data set generated from human cancer cell line OVCAR3.
Use: pFind; pParse; pGlyco
Molecular cell2018. Zhan, Ni et al.
Chinese Acad Sci, Inst Genet & Dev Biol, CAS Ctr Excellence Mol Plant Sci, Beijing 100101, Peoples R China; Chinese Acad Sci, State Key Lab Plant Genom, Beijing 100101, Peoples R China; Univ Chinese Acad Sci, Beijing 100049, Peoples R China
ABSTRACT:Nitric oxide (NO) regulates diverse cellular signaling through S-nitrosylation of specific Cys residues of target proteins. The intracellular level of S-nitrosoglutathione (GSNO), a major bioactive NO species, is regulated by GSNO reductase (GSNOR), a highly conserved master regulator of NO signaling. However, little is known about how the activity of GSNOR is regulated. Here, we show that S-nitrosylation induces selective autophagy of Arabidopsis GSNOR1 during hypoxia responses. S-nitrosylation of GSNOR1 at Cys-10 induces conformational changes, exposing its AUTOPHAGY-RELATED8 (ATG8)-interacting motif (AIM) accessible by autophagy machinery. Upon binding by ATG8, GSNOR1 is recruited into the autophagosome and degraded in an AIM-dependent manner. Physiologically, the S-nitrosylation-induced selective autophagy of GSNOR1 is relevant to hypoxia responses. Our discovery reveals a unique mechanism by which S-nitrosylation mediates selective autophagy of GSNOR1, thereby establishing a molecular link between NO signaling and autophagy.
Use: pFind
Journal of proteome research2018. Yang, Jing et al.
Wuhan Univ, Sch Pharmaceut Sci, Zhongnan Hosp, Wuhan 430071, Peoples R China; Tibet Univ, Coll Med, Lasa 850000, Peoples R China; Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China; Wuhan Univ, Sch Pharmaceut Sci, Key Lab Combinatorial Biosynth & Drug Discovery M, State Key Lab Virol, Wuhan 430071, Peoples R China; Wuhan Univ, Hubei Prov Key Lab Dev Originated Dis, Wuhan 430071, Peoples R China; Wuhan Univ, Sch Basic Med Sci, Hubei Prov Engn & Technol Res Ctr Fluorinated Pha, Ctr Expt Basic Med Educ, Wuhan 430071, Peoples R China; Anhui Med Univ, Hefei 230032, Anhui, Peoples R China
ABSTRACT:Targeted therapy of hepatocellular carcinoma (HCC) is essential for improved therapies. Therefore, identification of key targets specifically to HCC is an urgent requirement. Herein, an iTRAQ-quantitative proteomic approach was employed to identify differentially expressed proteins in HCC tumor tissues. Of the upregulated tumor-related proteins, minichromosome maintenance 2 (MCM2), a DNA replication licensing factor, was one of the most significantly altered proteins, and its over expression was confirmed using tissue microarray. Clinicopathological analysis of multiple cohorts of HCC patients indicated that overexpression of MCM2 was validated in 89.8% tumor tissues and strongly correlated with clinical stage. Furthermore, siRNA-mediated repression of MCM2 expression resulted in significant suppression of the HepG2 cell cycle and proliferation through the cyclin D-dependent kinases (CDKs) 2/7 pathway. Finally, the first small molecule-based MCM2-targeted NIR-II probe CH1055-MCM2 was concisely generated and subsequently evaluated in mice bearing HepG2 xenografts. The excellent imaging properties such as good tumor uptake and high tumor contrast and specificity were achieved in the small animal models. This analytical strategy can determine novel accessible targets of HCC useful for imaging and therapy.
Use: pFind
Nature Chemical Biology2018. Akter, S et al.
Scripps Res Inst, Dept Chem, Jupiter, FL 33458 USA.
ABSTRACT:Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1-4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species-associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.
Use: pFind; pQuant
2018. Fu, Ling et al.
ABSTRACT:
Use: pFind; pQuant
VIRUSES-BASEL2018. Aloor, A et al.
Georgia State Univ, Ctr Diagnost & Therapeut, Atlanta, GA 30302 USA.
ABSTRACT:Adeno associated virus (AAV) is a versatile gene delivery tool, which has been approved as a human gene therapy vector for combating genetic diseases. AAV capsid proteins are the major components that determine the tissue specificity, immunogenicity and in vivo transduction performance of the vector. In this study, the AAV8 capsid glycosylation profile was systemically analyzed by peptide mass fingerprinting utilizing high-resolution mass spectrometry to determine the presence of capsid glycosylation. We identified N-glycosylation on the amino acid N499 of the capsid protein. We characterized the overall sugar profile for vector produced in 293 cells. Multiple N-glycosylated host-cell proteins (HCPs) copurified with AAV8 vectors and were identified by analyzing LC-MS data utilizing a human database and proteome discoverer search engine. The N-glycosylation analysis by MALDI-TOF MS, highlighted the probability of AAV8 interaction with terminal galactosylated N-glycans within the HCPs.
Use: pFind
Structure2018. Karch, KR et al.
Univ Penn, Dept Biochem & Mol Biophys, Perelman Sch Med, Philadelphia, PA 19104 USA.
ABSTRACT:Until recently, a major limitation of hydrogen-deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization was limited to the length of the peptide generated during proteolysis. However, electron transfer dissociation (ETD) has been shown to preserve deuterium label in the gas phase, enabling better resolution. To date, this technology remains mostly limited to small, already well-characterized proteins. Here, we optimize, expand, and adapt HDX-MS tandem MS (MS/MS) capabilities to accommodate histone and nucleosomal complexes on top-down HDX-MS/MS and middle-down HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded analysis by traditional structural biology techniques for decades, revealing important insights into chromatin biology. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.
Use: pFind
Journal of Proteome Research2018. He, CT et al.
Anhui Med Univ, Hefei 230032, Anhui, Peoples R China.
ABSTRACT:Microproteins are peptides composed of 100 amino acids (AA) or fewer, encoded by small open reading frames (smORFs). It has been demonstrated that microproteins participate in and regulate a wide range of functions in cells. However, the annotation and identification of microproteins is challenging in part owing to their low molecular weight, low abundancy, and hydrophobicity. These factors have led to the unannotation of smORFs in genome processing and have made their identification at the protein level difficult. Large-scale enrichment of microproteins in proteogenomics has made it possible to efficiently identify microproteins and discover unannotated smORFs in Saccharomyces cerevisiae. We integrated four microprotein-specific enrichment strategies to enhance coverage. We identified 117 microproteins, verified 31 missing proteins (MPs), and discovered 3 novel smORFs. In total, 31 proteins were confirmed as MPs by spectrum quality checking. Three novel smORFs (YKL104W-A, YHR052C-B, and YHR054C-B) were reserved after spectrum quality checking, peptide synthesizing, homologue matching, and so on. This study not only demonstrates that there are potential smORF candidates to be annotated in an extensively studied organism but also presents an efficient strategy for the discovery of small MPs. All MS data sets have been deposited to the ProteomeXchange with identifier PXD008586.
Use: pFind
Molecular Plant2018. Yang, MK et al.
Chinese Acad Sci, Inst Hydrobiol, Key Lab Algal Biol, Wuhan 430072, Hubei, Peoples R China.
ABSTRACT:Diatoms comprise a diverse and ecologically important group of eukaryotic phytoplankton that significantly contributes to marine primary production and global carbon cycling. Phaeodactylum tricornutum is commonly used as a model organism for studying diatom biology. Although its genome was sequenced in 2008, a high-quality genome annotation is still not available for this diatom. Here we report the development of an integrated proteogenomic pipeline and its application for improved annotation of P. tricornutum genome using mass spectrometry (MS)-based proteomics data. Our proteogenomic analysis unambiguously identified approximately 8300 genes and revealed 606 novel proteins, 506 revised genes, 94 splice variants, 58 single amino acid variants, and a holistic view of post-translational modifications in P. tricornutum. We experimentally confirmed a subset of novel events and obtained MS evidence for more than 200 micropeptides in P. tricornutum. These findings expand the genomic landscape of P. tricornutum and provide a rich resource for the study of diatom biology. The proteogenomic pipeline we developed in this study is applicable to any sequenced eukaryote and thus represents a significant contribution to the toolset for eukaryotic proteogenomic analysis.
Use: pFind