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

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 (a glycan shield). The SARS-CoV-2 spike protein (S) has 22 potential sites of N-glycosylation per protein protomer. Here, mass spectrometric analysis of the N-glycosylation profiles of recombinant spike proteins from four Variants of Concern (Alpha, Beta, Gamma, and Delta) along with D614G as a control shows that amino acid substitutions and deletions between variants impact the abundance and type of glycans at spike glycosylation sites. While some sequons show variant-specific differences in glycan distributions, later variants showed high similarity in site-specific glycan content to S-D614G. Multiple digestion methods were applied per sample, and site-specific results were confirmed across different experimental conditions to improve glycopeptide identification and quantification. Detailed site-specific glycan analysis across variants informs the role of protein glycosylation in viral protein structure/function and supports development of vaccines and therapeutics.