New England Biolabs, Ipswich, MA, USA.
Methods Mol Biol. 2021;2271:273-280. doi: 10.1007/978-1-0716-1241-5_19.
The use of sequential exoglycosidase digestion of oligosaccharides followed by LC-FLD, LC-MS or CE analysis provides detailed carbohydrate structural information. Highly specific exoglycosidases cleave monosaccharides from the nonreducing end of an oligosaccharide and yield information about the linkage, stereochemistry and configuration of the anomeric carbon. Here we use combinations of exoglycosidases to precisely characterize glycans on the Fc domain of therapeutic antibodies and dimeric fusion proteins. The workflow described includes glycan release with Rapid™ PNGase F (NEB #P0710), direct labeling of released glycans with procainamide (PCA) or 2-aminobenzamide (2AB), cleanup of labeled glycans and a 3 h enzymatic digestion with exoglycosidases. This protocol is designed for completion within an 8 h time frame to allow for subsequent LC-FLD, LC-MS, or CE analysis overnight.
使用顺序外切糖苷酶消化寡糖，然后进行 LC-FLD、LC-MS 或 CE 分析，可以提供详细的碳水化合物结构信息。高度特异性的外切糖苷酶从寡糖的非还原端切割单糖，从而提供有关糖苷键、立体化学和端基碳原子构型的信息。在这里，我们使用外切糖苷酶组合来精确表征治疗性抗体和二聚融合蛋白 Fc 结构域上的聚糖。所描述的工作流程包括使用 Rapid™ PNGase F（NEB #P0710）进行聚糖释放，用普鲁卡因胺（PCA）或 2-氨基苯甲酰胺（2AB）直接标记释放的聚糖，标记聚糖的清洗，以及 3 小时的外切糖苷酶酶解。该方案设计在 8 小时内完成，以便在随后的 LC-FLD、LC-MS 或 CE 分析中过夜进行。
