Merry Anthony H, Neville David C A, Royle Louise, Matthews Brian, Harvey David J, Dwek Raymond A, Rudd Pauline M
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Anal Biochem. 2002 May 1;304(1):91-9. doi: 10.1006/abio.2002.5620.
The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60 degrees C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.
对糖蛋白O-连接聚糖进行定量分析的第一步是高产率、非选择性、无修饰且带有游离还原端地释放它们。与其他技术相比，肼解能够满足这些标准。然而，正如L. Royle等人在随附文章（2002年，《分析生物化学》第304期）中所描述的那样，在分析O-连接聚糖池时，观察到了一些聚糖的剥离现象。因此，通过分析和鉴定完整的O-聚糖及降解产物，对样品制备和聚糖回收的关键步骤进行了评估。通过质谱对合成的O-糖肽进行了表征。释放出的聚糖与糖肽上的聚糖相同。还分析了一系列复杂度不断增加的糖蛋白中的O-连接聚糖，即牛血清胎球蛋白、血型糖蛋白A以及从人唾液粘蛋白Muc5B中分离出的此前未表征的糖肽。聚糖谱的定量分析证实，在60摄氏度下肼解6小时并采用现在所描述的优化程序回收的条件下，O-连接聚糖的剥离率<2%。这表明可以通过肼解制备O-连接聚糖而不发生降解，并且作为分析策略的一部分，使得对低微克水平天然存在的糖蛋白上连接的O-连接聚糖的分析成为可能。
