title: Convenient and Precise Strategy for Mapping N-Glycosylation Sites Using Microwave-Assisted Acid Hydrolysis and Characteristic Ions Recognition.
abstract: N-glycosylation is one of the most prevalence protein post-translational modifications (PTM) which is involved in several biological processes. Alternation of N-glycosylation is associated with cellular malfunction and development of disease. Thus, investigation of protein N-glycosylation is crucial for diagnosis and treatment of disease. Currently, deglycosylation with peptide N-glycosidase F is the most commonly used technique in N-glycosylation analysis. Additionally, a common error in N-glycosylation site identification, resulting from protein chemical deamidation, has largely been ignored. In this study, we developed a convenient and precise approach for mapping N-glycosylation sites utilizing with optimized TFA hydrolysis, ZIC-HILIC enrichment, and characteristic ions of N-acetylglucosamine (GlcNAc) from higher-energy collisional dissociation (HCD) fragmentation. Using this method, we identified a total of 257 N-glycosylation sites and 144 N-glycoproteins from healthy human serum. Compared to deglycosylation with endoglycosidase, this strategy is more convenient and efficient for large scale N-glycosylation sites identification and provides an important alternative approach for the study of N-glycoprotein function.
authors.forename: Cheng
authors.initials: C
authors.lastname: Ma
authors.affiliation: §National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Jingyao
authors.initials: J
authors.lastname: Qu
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Meisner
authors.affiliation: ‡National Institute of Biological Sciences, Beijing 102206, People's Republic of China.
authors.forename: Xinyuan
authors.initials: X
authors.lastname: Zhao
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wu
authors.forename: Hailiang
authors.initials: H
authors.lastname: Zhu
authors.forename: Zaikuan
authors.initials: Z
authors.lastname: Yu
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Guo
authors.forename: Jing
authors.initials: J
authors.lastname: Song
authors.affiliation: §National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, People's Republic of China.
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis / Q000097:blood* / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007477:Ions / Q000032:analysis*; D013058:Mass Spectrometry; D008872:Microwaves; D015394:Molecular Structure
chemical_list: D015415:Biomarkers; D006023:Glycoproteins; D007477:Ions