Confident characterization of the micro-heterogeneity 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.
Prof. Catherine C.L. Wong’s lab, in National Center for Protein Science·Shanghai, Institute of Biochemistry and Cell Biology (SIBCB), Institutions of Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences(CAS) together with their collaborators, Prof. Peng-yuan Yang’s group in Institutes of Biomedical Sciences, Fudan University and Prof. Si-min He’s group in Key Lab of Intelligent information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, proposed a novel pipeline(pGlyco) 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.
Dr. Chao Peng in National Center for Protein Science-Shanghai provided technical support and collected the high quality MS data. The Protein Science Center is equipped with the state-of-the-art facilities and enabling technologies for life science study and provides superb service for domestic and international users.
This work entitled “pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD and CID-MS/MS and MS3”, was published in Scientific Reports on May 3TH, 2016.
This work was supported by grants from the National Basic Research Program of China, National Natural Science Foundation of China project, Hi-Tech Research and Development Program of China (863 grants).
Mass spectrometry system has been providing full and high quality support for a number of national science and technology strategic pilot projects, such as 973 and 863. It has been serving more than 250 client projects since May 2014. Since then, it has supported scientists to publish more than 10 research articles in high impact factor peers reviewed journal such as Science, Nature, Nature journals, Mol Cell, PNAS, JACS, etc. It contributed as co-corresponding author on two papers published on EMBOJ and Scientific Report, respectively.
AUTHOR CONTACT
Catherine C.L. Wong Ph.D. Professor
National center for protein science(Shanghai),Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
333 Haike Road, Shanghai 201210, China
Tel: 86-21-20778066
Email: catherine_wong@sibcb.ac.cn
The overall workflow of pGlyco
(Image provided by Prof. Catherine C.L. Wong’s lab)
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