Site-specific N- and O-glycosylation Analysis of Human Plasma Fibronectin

Human plasma fibronectin is an adhesive protein that plays a crucial role in wound healing. Many studies had indicated that glycans may 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-glycosites 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 3842 N-glycoforms and 16 O-glycoforms, respectively. Furthermore, 81.4% of N-glycans were either fucosylated, sialylated, or with both modifications77.31% of N-glycans were sialylated, while 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.