• Identified 15 O-glycosites and 10 distinct O-glycan structures on the SARS-CoV-2 spike protein using advanced mass spectrometry.
• Demonstrated that ppGalNAc-T6 is a key host enzyme enhancing O-glycosylation of the spike protein, increasing both site occupancy and glycan heterogeneity.
• Molecular dynamics simulations revealed that O-glycosylation at protomer interfaces stabilizes the trimeric spike structure via hydrogen bonds and non-polar interactions.
• Conservation analysis suggests that most O-glycosites are maintained across SARS-CoV-2 variants, highlighting their potential functional importance.