title: pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3.
abstract: Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification; 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides.
authors.affiliation: Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Wen-Feng
authors.initials: WF
authors.lastname: Zeng
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Ming-Qi
authors.initials: MQ
authors.lastname: Liu
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Jian-Qiang
authors.initials: JQ
authors.lastname: Wu
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Fang
authors.affiliation: National Center for Protein Science (Shanghai), Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Peng
authors.affiliation: Thermo Fisher Scientific Co., Ltd, Shanghai, China.
authors.forename: Aiying
authors.initials: A
authors.lastname: Nie
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Weiqian
authors.initials: W
authors.lastname: Cao
authors.affiliation: Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.affiliation: Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chi
authors.affiliation: Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.
authors.forename: Rui-Xiang
authors.initials: RX
authors.lastname: Sun
authors.affiliation: National Center for Protein Science (Shanghai), Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Catherine C L
authors.initials: CC
authors.lastname: Wong
authors.affiliation: Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.|University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Si-Min
authors.initials: SM
authors.lastname: He
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
mesh_terms: D006020:Glycopeptides / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D057188:Workflow
chemical_list: D006020:Glycopeptides