Determining Site-Specific Glycan Profiles of Recombinant SARS-CoV‑2 Spike Proteins from Multiple Sources

Glycopeptide Abundance Distribution Spectra (GADS) were
recently
introduced as a means of representing, storing, and comparing glycan
profiles of intact glycopeptides. Here, using that representation,
an extensive analysis is made of multiple commercial sources of the
recombinant SARS-CoV-2 spike protein, each containing 22 N-linked
glycan sites (sequons). Multiple proteases are used along with variable
energy fragmentation followed by ion trap confirmation. This enables
a detailed examination of the reproducibility of the method across
multiple types of variability. These results show that GADS are consistent
between replicates and laboratories for sufficiently abundant glycopeptides.
Derived GADS enable the examination and comparison of the glycan profiles
between commercial sources of the spike protein. Multiple distinct
glycopeptide distributions, generated by multiple proteases, confirm
these profiles. Comparisons of GADS derived from 11 sources of recombinant
spike protein reveal that sources for which protein expression methods
were the same produced near-identical glycan profiles, thereby demonstrating
the ability of this method to measure GADS of sufficient reliability
to distinguish different glycoform distributions between commercial
vendors and potentially to reliably determine and compare differences
in glycosylation for any glycoprotein under different conditions of
production. All mass spectrometry data files have been deposited in
the MassIVE repository under the identifier MSV000091776.