Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin.
Site-Specific N- and O-Glycosylation Analysis of Human Plasma Fibronectin.
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DOI：
10.3389/fchem.2021.691217
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发表时间：
2021
影响因子：
5.5
通讯作者：
Li L
中科院分区：
文献类型：
--
作者：
Liu D;Wang S;Zhang J;Xiao W;Miao CH;Konkle BA;Wan XF;Li L
人血浆纤连蛋白是一种粘附蛋白，在伤口愈合中起着至关重要的作用。许多研究表明，糖基化可能介导纤维连接蛋白的表达和功能，但对其糖基化的认识还不全面。在这里，我们进行了一个全面的N-和O-糖基化映射的人血浆纤连蛋白和量化的发生，每一个糖型的位点特异性的方式。完整的N-糖肽通过两性离子亲水相互作用色谱富集，N-糖位点通过18 O-标记方法定位。通过酶辅助定点提取法实现O-糖肽富集和O-糖基鉴定。采用碰撞诱导解离-阶梯归一化碰撞能（sNCE）-HCD串联质谱（RP-LC-MS/MS）技术对纤维连接蛋白的糖型进行分析。共鉴定出6个N-糖位点和53个O-糖位点，分别被38个N-糖型和16个O-糖型占据。此外，77.31%的N-聚糖被唾液酸化，并且O-糖基化由唾液酸化-T抗原主导。这些人纤连蛋白上的位点特异性糖基化模式可以促进纤连蛋白的功能分析和治疗药物的开发。
Human 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.