Glycosylation is one of these chemical modifications strategies and has shown significant promise in enhancing the proteolytic stability and monomeric propensity of various kinds of proteins . While N-linked glycosylation has been more studied due to better controllability and the ease of chemical synthesis, O-linked glycosylation has a larger diversity of carbohydrate structures and potentially a greater ability for tuning biophysical properties of glycoproteins. Guan et al. systematically investigated different glycosylation patterns, including glycosylation sites, glycan sizes, and glycan structures . Their study suggested the superiority of O-mannosylation of insulin B-chain Thr27 to other studied glycosylation patterns in increasing the proteolytic stability against 𝛼-chymotrypsin and decreasing the dimerization propensity of insulin, while still maintaining full biological activity. However, there is a lack of quantitative understanding of how insulin properties are influenced by the interactions between the attached sugar molecules and insulin residues, particularly at the molecular level.