Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2 play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we have utilized individual ion mass spectrometry (I2MS), a form of charge detection mass spectrometry (CDMS) that uses a commercially available Orbitrap analyzer, to directly produce heterogeneous glycan mass profiles of these three coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques. This highlights the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts and differences in glycan removal are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional protein mass analysis methods such as size exclusion chromatography-multiple angle light scattering (SEC-MALS) and field flow fractionation-multiple angle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.