Hierarchy of N-acylation sites in human insulin studied by RP-HPLC and mass spectrometry

Acylation of human insulin was studied using,c2 and c12 transfer reagents,nsuccinimidyl acetate and n-succinimidyl dodecanoate (lauryl) respectively. in general,the acylation reaction was more specific at high ph values (12.00) for the modification of the ε-amino group of k29b. at ph around 8.5,multiple modifications occurred at other sites,in particular the n-terminal glycine of a-chain (g1a). withn-succinimidyl dodecanoate,about 88 % of the acylation was established to be at the ε-amino of k29b,as shown using tryptic digestion and identification of the products by mass spectrometry. while with n-succinimidyl acetate,singly acetylated product with acetyl group at the ε-amino of k29b,as well as doubly modified species containing acetyl group in the ε-amino group and the amino-terminal of g1a were produced in equal amounts. the high regiospecificity of modification at the ε-amino group,by the c12 reagent,is attributed to steric factors. moreover,the above investigation also highlights the purification regime of singly and doubly acetylated products on rp-hplc using a shallow gradient,though the difference between two species,in the case of acetyl group,is of 43 da. the overall results allow us to establish the hierarchy of accessibility of the three amino group; ε-amino group k29b is the least hindered and accessible to c2 as well as c12 reagents,then is the amino group of g1a which is accessible to the c2 but not the c12 reagent,finally that of f1b is most hindered and accessible to neither.