Liu, DingWang, ShuaishuaiZhang, JunpingXiao, WeidongMiao, Carol H.Konkle, Barbara A.Wan, Xiu-FengLi, Lei2024-04-012024-04-012021-06-15Liu D, Wang S, Zhang J, et al. Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin. Front Chem. 2021;9:691217. Published 2021 Jun 15. doi:10.3389/fchem.2021.691217https://hdl.handle.net/1805/39650Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans might mediate the expression and functions of fibronectin, yet a comprehensive understanding of its glycosylation is still missing. Here, we performed a comprehensive N- and O-glycosylation mapping of human plasma fibronectin and quantified the occurrence of each glycoform in a site-specific manner. Intact N-glycopeptides were enriched by zwitterionic hydrophilic interaction chromatography, and N-glycosite sites were localized by the 18O-labeling method. O-glycopeptide enrichment and O-glycosite identification were achieved by an enzyme-assisted site-specific extraction method. An RP–LC–MS/MS system functionalized with collision-induced dissociation and stepped normalized collision energy (sNCE)-HCD tandem mass was applied to analyze the glycoforms of fibronectin. A total of 6 N-glycosites and 53 O-glycosites were identified, which were occupied by 38 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 77.31% of N-glycans were sialylated, and O-glycosylation was dominated by the sialyl-T antigen. These site-specific glycosylation patterns on human fibronectin can facilitate functional analyses of fibronectin and therapeutics development.en-USAttribution 4.0 InternationalFibronectinGlycosylationMass spectrometerOperatorStepped normalized collision energyArticle