Figure shows the percentage of the time each kind of hydrogen bond existed in the MD simulations. As a result, proteolytically stable glycoforms such as GF 13 and GF 10 had the most glycan-involved hydrogen bonds. Glycoforms with comparable proteolytic stability as the wild type, including GF 5, GF9, and GF 12 had at least one or two glycan-involved hydrogen bonds. Most of the remaining glycoforms, which were more susceptible to proteolytic degradation than the wild type, had no glycan-involved hydrogen bonds. Importantly, glycoforms having at least comparable proteolytic stability as the wild type typically have hydrogen bonds that involved residues PheB24 or ThrB27, which were the P1 site and the P2' site corresponding to the cleavage site B25, respectively. In glycoforms that had an intermediate level of proteolytic stability (GF 5, and GF 9), hydrogen bonds involving these two residues were not present or at least not long-lasting in the MD trajectories (see Supplemental Table ), as reflected by the large errors bars that almost reached the bottom of the graph. On the other hand, in the proteolytically stable glycoforms, GF 13 and GF 10, hydrogen bonds ThrB27(N)-Man (O6) and PheB24(N)-Man (O3) were found in all GF 13/GF 10 MD trajectories, regardless of which wild-type model the glycoform was built on. We concluded these two kinds of hydrogen bonds (ThrB27(N)-Man (O6) and PheB24(N)-Man (O3)), the longest-lasting glycan-involved hydrogen bonds in the most proteolytically stable structures, were the most important hydrogen bonds influencing the proteolytic stability in our study. Interestingly, GF 13 occasionally had a hydrogen bond formed between ThrB27 and the third mannose, which was absent from GF 10. This additional hydrogen bond that involved a P2' site might further interfere with the hydrogen bonding network between insulin and 𝛼-chymotrypsin, making GF 13 even more proteolytically stable than GF 10.