In this study, we aim to identify structural determinants from MD simulations of glycosylated insulin that lead to enhanced proteolytic stability against 𝛼-chymotrypsin or reduced dimerization propensity. Specifically, we performed molecular dynamics simulations for each of the 12 insulin glycoforms (GFs) (Figure ) studied in the experimental work done by Guan et al. Each of the glycoforms was built upon five different wild-type models resolved by different methods/groups to encapsulate a wider variety of initial configurations so as to sample the conformational ensemble more comprehensively. Based on the proteolytic degradation mechanism by 𝛼-chymotrypsin and the structural characteristics of insulin, we proposed several potential metrics for assessing the two insulin properties, including solvent-accessible surface area (SASA), secondary structures, the existence of glycan-involved hydrogen bonds, and the occlusion between the glycan and the dimer interface. A previous study found that greater conformational rigidity of a carbohydrate binding module (CBM) protein was associated with larger proteolytic stability of glycoforms, thus disfavoring reaction-capable transition states. However, the overall conformational rigidity of insulin is significantly different from CBM, and thus more specific measures of transition state accessibility were used, such as the 𝛽-sheet propensity, which more specifically takes the structural features of the known 𝛼-chymotrypsin transition state into account. (blue), ThrB27 (orange) and ThrB30 (green) all represented as beads. The glycosylation pattern of each glycoform is implied by its name-for example, GalNAc𝛼 (SerA9) represents the glycoform having an N-acetylgalactosamine (GalNAc) attached to the A chain Ser9 residue. Note that for glycoforms having a mannose dimer or trimer as the glycan, the linkages between monomers were all 𝛼-1,2-linkages, so Tri-Man𝛼 (ThrB27) refers to the glycoform containing an 𝛼-1,2linked tri-mannose at the B chain Thr27 residue.