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.
Keywords: CE; Exoglycosidase; Glycan Structure; LC-FLD; LC-MS; Rapid™ PNGase F.