title: Proteomics Analysis of O-GalNAc Glycosylation in Human Serum by an Integrated Strategy.
abstract: The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Hongqiang
authors.identifier: 0000-0002-7508-0872
authors.initials: H
authors.lastname: Qin
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Mao
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Fangjun
authors.identifier: 0000-0002-8118-7019
authors.initials: F
authors.lastname: Wang
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Dong
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Zhimou
authors.initials: Z
authors.lastname: Guo
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Xinmiao
authors.identifier: 0000-0001-5802-1961
authors.initials: X
authors.lastname: Liang
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.affiliation: CAS Key Lab of Separation Science for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D060748:Fetuins / Q000378:metabolism; D006020:Glycopeptides / Q000097:blood*; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D001798:Blood Proteins; D060748:Fetuins; D006020:Glycopeptides