title: Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides.
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 in18O 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. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples. Graphical abstract Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.
authors.affiliation: Anhui Medical University, No. 81 Meishan Road, Hefei, Anhui, 230032, China.|National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Zixiang
authors.initials: Z
authors.lastname: Yu
authors.affiliation: National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Zhao
authors.affiliation: National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Tian
authors.affiliation: National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Huang
authors.affiliation: National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Qian
authors.affiliation: Anhui Medical University, No. 81 Meishan Road, Hefei, Anhui, 230032, China. proteomics@126.com.|National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China. proteomics@126.com.
authors.forename: Wantao
authors.initials: W
authors.lastname: Ying
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D056945:Hep G2 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012260:Ribonucleases / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006020:Glycopeptides; D007074:Immunoglobulin G; D012260:Ribonucleases; C029600:ribonuclease B; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Byonic; Glycoprotein; Glycosylation; HILIC; Mass spectrometry