Hydrogen-Deuterium Exchange Epitope Mapping of Glycosylated Epitopes Enabled by Online Immobilized Glycosidase

Hydrogen-deuterium exchange coupled with massspectrometry(HDX-MS) is widely used for monoclonal antibody (mAb) epitopemapping, which aids in the development of therapeutic mAbs and vaccines,as well as enables the understanding of viral immune evasion. NumerousmAbs are known to recognize N-glycosylated epitopes and to bind inclose proximity to an N-glycan site; however, glycosylatedprotein sites are typically obscured from HDX detection as a resultof the inherent heterogeneity of glycans. To overcome this limitation,we covalently immobilized the glycosidase PNGase Dj on a solid resinand incorporated it into an online HDX-MS workflow for post-HDXdeglycosylation. The resin-immobilized PNGase Dj exhibited robusttolerance to various buffer conditions and was employed in a columnformat that can be readily adapted into a typical HDX-MS platform.Using this system, we were able to obtain full sequence coverage ofthe SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylatedepitope of the glycan-binding mAb S309 to the RBD.

Automated summary

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is used for epitope mapping of monoclonal antibodies (mAb) and understanding viral immune evasion. To overcome the limitation of glycan heterogeneity, a glycosidase was covalently immobilized on a resin and incorporated into an online HDX-MS workflow for post-HDX deglycosylation. This system allowed for full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and mapping of the glycosylated epitope of a glycan-binding mAb S309.