Analysis of the N-glycosylation profiles of the spike proteins from the Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2

Anal Bioanal Chem. 2023 Aug;415(19):4779-4793. doi: 10.1007/s00216-023-04771-y. Epub 2023 Jun 24.


N-Glycosylation plays an important role in the structure and function of membrane and secreted proteins. Viral proteins used in cell entry are often extensively glycosylated to assist in protein folding, provide stability, and shield the virus from immune recognition by its host (described as a "glycan shield"). The SARS-CoV-2 spike protein (S) is a prime example, having 22 potential sites of N-glycosylation per protein protomer, as predicted from the primary sequence. In this report, we conducted mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins derived from four common SARS-CoV-2 variants classified as Variant of Concern, including Alpha, Beta, Gamma, and Delta along with D614G variant spike as a control. Our data reveal that the amino acid substitutions and deletions between variants impact the abundance and type of glycans on glycosylation sites of the spike protein. Some of the N-glycosylation sequons in S show differences between SARS-CoV-2 variants in the distribution of glycan forms. In comparison with our previously reported site-specific glycan analysis on the S-D614G and its ancestral protein, glycan types on later variants showed high similarity on the site-specific glycan content to S-D614G. Additionally, we applied multiple digestion methods on each sample, and confirmed the results for individual glycosylation sites from different experiment conditions to improve the identification and quantification of glycopeptides. Detailed site-specific glycan analysis of a wide variety of SARS-CoV-2 variants provides useful information toward the understanding of the role of protein glycosylation on viral protein structure and function and development of effective vaccines and therapeutics.

Keywords: Mass spectrometry; N-Glycosylation; SARS-CoV-2 spike variants.

MeSH terms

  • COVID-19*
  • Glycosylation
  • Humans
  • Polysaccharides / chemistry
  • SARS-CoV-2* / genetics
  • SARS-CoV-2* / metabolism
  • Spike Glycoprotein, Coronavirus / chemistry


  • spike protein, SARS-CoV-2
  • Spike Glycoprotein, Coronavirus
  • Polysaccharides

Supplementary concepts

  • SARS-CoV-2 variants