Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry

► Mass spectrometry quantified hierarchy of tryptic sites in proinsulin derivatives. ► Km, kcat., and kcat./Km were obtained for tryptic sites in proinsulin derivatives. ► K29-T30 tryptic cleavage shielded in proinsulin/insulin, not in di-arginyl insulin. ► B/C site variation of human and buffalo proinsulin causes tryptic regioselectivity.