Hydrogen deuterium exchange epitope mapping of glycosylated epitopes enabled by online immobilized glycosidase

Numerous mAbs are known to recognize N-glycosylated epitopes or to bind in close proximity to an N-glycan site, however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibits a robust tolerance to various buffer conditions, and is employed in a column format that can be readily adapted into a typical HDX-MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor binding domain (RBD) and to map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.