Enabling Epitope Analysis of Glycoproteins via HDX-MS Using Antigens with Uni-formed N-linked Glycans

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is widely used for epitope analysis of the antibod-ies. However, epitope analysis of glycoproteins is challenging because of the heterogeneity of attached N-linked glycans. Recent studies have reported methods where N-linked glycans were removed at low pH follow-ing hydrogen deuterium exchange reactions, but the methods for glycoproteins remain controversial. Here, we demonstrate the utility of using antigens with uniformed N-linked glycans in glycoprotein epitope analysis by HDX-MS. By treating HEK293 cells with kifunensine, we were able to prepare antigens mainly with N-linked high-mannose-type glycans. Analysis of epitopes of a monoclonal antibody S309 using antigens prepared with this method allowed us to identify an epitope that included the previously reported N-linked glycan attachment sites for this antibody. We propose that using antigens with uniformed glycans should be effective for epitope analysis of glycoproteins, such as virus spike proteins covered by glycan shields. Moreover, we believe that this approach accelerates research on vaccines and neutralizing antibodies against viruses that escape host immunity through glycan shields or mutations.