Ludger Ltd., Culham Science Centre, Oxfordshire OX14 3EB, UK.
Anal Biochem. 2012 Apr 1;423(1):119-28. doi: 10.1016/j.ab.2012.01.002. Epub 2012 Jan 20.
The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method.
O-聚糖分析对于更好地理解它们在生物过程中的功能至关重要。尽管已经开发出许多 O-聚糖释放技术，但肼解释放方法是高产率产生具有游离还原末端的 O-聚糖的最佳方法。这种释放技术允许糖被标记上荧光团，并通过荧光检测进行分析。在肼解释放条件下，观察到副反应，导致聚糖还原末端的单糖损失（称为剥皮）。使用牛胎球蛋白（因为它含有生物制药中最常见的唾液酸化 O-聚糖）和牛颌下腺粘蛋白（BSM），我们证明在肼解之前，用 0.1%三氟乙酸（TFA）或低摩尔浓度（100、50、20 和 5 mM）乙二胺四乙酸（EDTA）溶液进行缓冲交换，可以大大减少剥皮。氯化钙的添加导致胎球蛋白的剥皮增加，而随后用 EDTA 洗涤则消除了这种影响，表明钙和可能的其他阳离子在引起剥皮中起作用。在肼解之前进行样品制备的所提出的技术大大降低了 O-聚糖分析中不期望的裂解产物的水平，并提高了该方法的稳健性。
