Rosner M R, Robbins P W
J Cell Biochem. 1982;18(1):37-47. doi: 10.1002/jcb.1982.240180105.
A method is presented for separation of tryptic glycopeptides-containing oligosaccharides of the N-asparagine-linked type. High performance liquid chromatography (HPLC) of glycopeptides on a C18 reverse-phase system eluted with a gradient of 0%-50% acetonitrile in 0.1 M NaPO4 pH 2.2 resolves the two major glycosylation sites from the envelope glycoprotein (G) of vesicular stomatitis virus. Glycopeptides containing N-linked oligosaccharides of the complex type coelute with those containing N-linked oligosaccharides of the neutral, high mannose type, indicating that separation is based upon peptide rather than carbohydrate composition. The contribution of the carbohydrate component to glycopeptide elution, as determined by cleavage of the high mannose oligosaccharides with endo-beta-Nacetylglucosaminidase H, is that of a significant, but minor, decrease in peptide retention time. Comparison of the tryptic glycopeptide profiles of G isolated from both wild type and mutant strains of VSV illustrates the rapid, reproducible, and quantitative nature of the technique. Through HPLC analysis of appropriately treated glycopeptides, it is possible to explore both the nature and extent of glycosylation at individual sites in glycoproteins in a single step.
本文介绍了一种分离含N - 天冬酰胺连接型寡糖的胰蛋白酶糖肽的方法。在C18反相系统上，用0.1 M NaPO4（pH 2.2）中0% - 50%乙腈梯度洗脱糖肽的高效液相色谱（HPLC）法，可分离水疱性口炎病毒包膜糖蛋白（G）的两个主要糖基化位点。含复合型N - 连接寡糖的糖肽与含中性、高甘露糖型N - 连接寡糖的糖肽共洗脱，这表明分离是基于肽而非碳水化合物组成。用内切β - N - 乙酰葡糖胺糖苷酶H切割高甘露糖寡糖来确定碳水化合物成分对糖肽洗脱的贡献，结果是肽保留时间显著但轻微减少。对从水疱性口炎病毒野生型和突变株分离的G的胰蛋白酶糖肽图谱进行比较，说明了该技术的快速、可重复和定量性质。通过对经过适当处理的糖肽进行HPLC分析，有可能在一步中探索糖蛋白中各个位点糖基化的性质和程度。
