Ezose Sciences, Inc., 25 Riverside Drive Pine Brook, New Jersey 07058, United States.
Anal Chem. 2010 Dec 15;82(24):10021-9. doi: 10.1021/ac101599p. Epub 2010 Nov 15.
Glycoblotting, high throughput method for N-glycan enrichment analysis based on the specific chemical ligation between aminooxy/hydrazide-polymers/solids and reducing N-glycans released from whole serum and cellular glycoproteins, was proved to be feasible for selective enrichment analysis of O-glycans of common (mucin) glycoproteins. We established a standard protocol of glycoblotting-based O-glycomics in combination with nonenzymatic chemical treatment to release reducing O-glycans predominantly from various glycoprotein samples. It was demonstrated that the nonreductive condition employing a simple ammonium salt, ammonium carbamate, made glycoblotting-based enrichment analysis of O-glycans possible without significant loss or unfavorable side reactions. A general workflow of glycoblotting using a hydrazide bead (BlotGlyco H), on-bead chemical manipulations, and subsequent mass spectrometry allowed for rapid O-glycomics of human milk osteopontin (OPN) and urinary MUC1 glycoproteins purified from healthy donors in a quantitative manner. It was revealed that structures of O-glycans in human milk OPN were varied with habitual fucosylation and N-acetyllactosamine units. It was also suggested that purified human urinary MUC1 was modified preferentially by sialylated O-glycans (94% of total) with 7:3 ratio of core 1 to core 2 type O-glycans. Versatility of the present strategy is evident because this method was proved to be suited for the enrichment analysis of general biological and clinical samples such as human serum and urine, cultured human cancer cells, and formalin-fixed paraffin-embedded tissue sections. It is our belief that the present protocols would greatly accelerate discovery of disease-relevant O-glycans as potential biomarkers.
糖基印迹法是一种基于氨基氧基/酰肼聚合物/固体与从全血清和细胞糖蛋白中释放的还原 N-聚糖之间的特异性化学连接的高通量 N-聚糖富集分析方法，已被证明可用于常见（粘蛋白）糖蛋白的 O-聚糖的选择性富集分析。我们建立了一种基于糖基印迹的 O-糖组学标准方案，结合非酶化学处理，主要从各种糖蛋白样品中释放还原 O-聚糖。结果表明，采用简单的铵盐——碳酸铵，在非还原条件下，可以进行基于糖基印迹的 O-聚糖富集分析，而不会有明显的损失或不利的副反应。使用酰肼珠（BlotGlyco H）进行糖基印迹、珠上化学操作以及随后的质谱分析的一般工作流程，可以快速定量分析人乳骨桥蛋白（OPN）和健康供体尿液 MUC1 糖蛋白的 O-糖组学。结果表明，人乳 OPN 中的 O-聚糖结构因习惯性岩藻糖基化和 N-乙酰乳糖胺单位而有所不同。研究还表明，纯化的人尿 MUC1 主要被唾液酸化的 O-聚糖修饰（占总糖的 94%），核心 1 型和核心 2 型 O-聚糖的比例为 7:3。本研究策略的通用性显而易见，因为该方法已被证明适用于人血清和尿液、培养的人类癌细胞以及福尔马林固定石蜡包埋组织切片等一般生物和临床样本的富集分析。我们相信，本研究方案将极大地加速疾病相关 O-聚糖作为潜在生物标志物的发现。
