Heterogeneity of Glycan Processing on Trimeric SARS-CoV-2 Spike Protein Revealed by Charge Detection Mass Spectrometry

The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by top-down CDMS measurements are 35-47% larger than those obtained from the bottom-up glycoproteomics studies, suggesting that the glycoproteomic measurements under- estimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host's immune system.

Automated summary

The heterogeneity of glycosylation on the protein trimer of SARS-CoV-2 virus has been evaluated using charge detection mass spectrometry (CDMS), showing a broad distribution of glycan processing. CDMS measurements reveal significantly larger average glycan masses than those obtained from glycoproteomics studies, indicating an underestimation of the abundances of larger, more complex glycans.