Characterization of O-glycans linked to serine or threonine residues in glycoproteins has mostly been achieved using chemical reaction approaches because there are no known O-glycan-specific endoglycosidases. Most O-glycans are modified with sialic acid residues at the non-reducing termini through various linkages. In this study, we developed a novel approach for sialic acid linkage-specific O-linked glycan analysis through lactone-driven ester-to-amide derivatization combined with non-reductive β-elimination in the presence of hydroxylamine. O-glycans released by non-reductive β-elimination were efficiently purified using glycoblotting via chemoselective ligation between carbohydrates and a hydrazide-functionalized polymer, followed by modification of methyl or ethyl ester groups of sialic acid residues on solid-phase. In-solution lactone-driven ester-to-amide derivatization of ethyl-esterified O-glycans was performed, and the resulting sialylated glycan isomers were discriminated by mass spectrometry. In combination with PNGase F digestion, we carried out simultaneous, quantitative, and sialic acid linkage-specific N- and O-linked glycan analyses of a model glycoprotein and human cartilage tissue. This novel glycomic approach will facilitate detailed characterization of biologically relevant sialylated N- and O-glycans on glycoproteins.

与糖蛋白中的丝氨酸或苏氨酸残基相连的O-聚糖的表征主要是使用化学反应方法实现的，因为没有已知的O-聚糖特异性内切糖苷酶。大多数O-聚糖在非还原末端通过各种连接被唾液酸残基修饰。在这项研究中，我们开发了一种新方法，用于通过内酯驱动的酯-酰胺衍生化结合非还原性 β-消除在羟胺存在下分析唾液酸键特异性 O-连接聚糖。欧通过碳水化合物和酰肼官能化聚合物之间的化学选择性连接，使用糖印迹法有效纯化非还原性 β- 消除释放的 - 聚糖，然后在固相上修饰唾液酸残基的甲基或乙基酯基团。进行了乙酯化O-聚糖的溶液内酯驱动的酯-酰胺衍生化，并通过质谱法区分了所得的唾液酸化聚糖异构体。结合 PNGase F 消化，我们进行了同步、定量和唾液酸键特异性N - 和O-模型糖蛋白和人类软骨组织的连接聚糖分析。这种新颖的糖组学方法将有助于详细表征糖蛋白上与生物学相关的唾液酸化N - 和O - 聚糖。