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

Abstract

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 underestimated 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.

Probing Affinity, Avidity, Anti-Cooperativity, and Competition in Antibody and Receptor Binding to the SARS-CoV-2 Spike by Single Particle Mass Analyses

“…43 The calculated glycan (92.0 kDa) and resultant total S-trimer (482.4 kDa) masses agree quite well (within 2%) with the masses measured by both MP and CD-MS. The glycan mass contribution measured here is somewhat lower than the recent results of Miller and co-workers 44 who reported large mass discrepancies of ~40% from similar glycoproteomic experiments. However, it should be noted the constructs used in that study differ from the one employed here in several key aspects (e.g.…”

Discrepancies between High-Resolution Native and Glycopeptide-Centric Mass Spectrometric Approaches: A Case Study into the Glycosylation of Erythropoietin Variants

“…Studying the effects of other proteases would be warranted. Of note, during the peer review period of this study, Miller et al 29 published the characterization of intact trimeric SARS-CoV-2 spike protein by charge detection mass spectrometry. When comparing their results to the glycopeptide studies utilizing trypsin, chymotrypsin, or alpha lytic protease, it was revealed that average glycan masses obtained by their intact approach are up to 47% larger than those reported by glycoproteomics studies.…”

The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor

“…Indeed, the polypeptide chain contains nearly two dozen N-glycosylation sites [ 20 – 22 ], and while the initial reports identified relatively few O-glycosylation sites [ 22 ], the latter have also been shown to be numerous in recent literature [ 23 ]. The large number of the glycosylation sites and the microheterogeneity at each site give rise to a broad distribution of the total carbohydrate mass, which in the case of the trimeric S-protein was estimated to span a 100–200-kDa range [ 24 ]. Such an enormous degree of structural heterogeneity is a serious challenge vis-à-vis extracting meaningful information on protein interactions from the native MS data.…”