Inclusion of glycopeptides in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike ectodomain provides in-creased sequence coverage

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein’s conformational dynamics across varied states, such as heat-denatured vs. native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of glycopeptides has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their isotopic mass shifts from deuteration. Inclusion of the deu-terated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. Abstract Figure