Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

“…Subtracting the sequence mass (134.4 kDa) from the monomer mass (162 kDa) yields an average glycan mass of 27.6 kDa, which is much smaller than the corresponding glycan masses for the spike monomers from HEK293 cells (61.4 kDa) and CHO cells (52.4 kDa). This observation is consistent with the conclusions of Zhang et al, who found the glycans from the S protein expressed in insect cell lines were mainly of the high mannose type while glycans from mammalian cell lines were mainly complex . Thus, the lower mass for the spike protein derived from insect cell expression is a result of different processing of the glycans in insect cells versus mammalian cells.…”

“…Stemming from the unreliability of X-ray crystallography and Cryo-EM techniques to spot glycosylated portions of the SARS-CoV-2 framework, the Crispin group adopted a site-specific mass spectrometric approach to reveal all monomeric N-linked glycan sequons per protomer of the S protein. Similar results were obtained by Yang's group, 36 which used a stepped collision energy mass spectrometry approach that revealed the Nglycosylation outline of the viral S proteins along with the glycan composition and site-specific numbers of glycans. In parallel, Azadi et al engaged the proficiency of high-resolution liquid chromatography-tandem mass spectrometry to identify not only 17 out of the predicted 22 N-linked glycans sites, but serendipitously also two O-linked glycans out of the four predicted sites.…”

“…With 23, 23, and 22 N-linked glycan sites per monomer for MERS, SARS-CoV, and SARS-CoV-2, respectively, extreme heterogeneity is expected within each spike protein trimer, as has been confirmed in the literature from analytical characterization of these proteins. ,,,,− This heterogeneity is also demonstrated by the broad mass profiles observed from I 2 MS measurements, with fwhm values of 14.6, 14.0, and 13.4 kDa, respectively, for MERS, SARS-CoV, and SARS-CoV-2 spike protein trimers. While these fwhm values alone are only an indicator of glycan heterogeneity, we also used previously published site-specific glycan structure results from bottom-up glycopeptide analysis of these same three spike protein trimers , to interrogate the theoretical distribution of protein mass for each spike protein using a Monte Carlo approach, similar to that previously published .…”