Comprehensive Characterization of Disulfide Bonds in Insulin and Its Metabolites from Liver S9 by a Proteomic Method

Disulfide bonds are essential for the structural and functional integrity of therapeutic peptides and proteins (TPPs). However, accurately mapping disulfide bonds in TPPs remains challenging due to the scrambling of the disulfide bond during sample preparation. In addition, identifying TPP metabolites with disulfide bonds is crucial for preclinical and clinical development. However, existing software for metabolite identification can only identify metabolites with native disulfide bonds, despite the evidence of nonnative disulfide bonds in metabolites. This highlights the necessity for developing a strategy for comprehensively profiling the metabolites of TPPs. In this study, a proteomic strategy based on pLink software was developed for the characterization of disulfide bonds in insulin and its metabolites from rat liver S9. Through peptic digestion at 0 °C and subsequent liquid chromatography–high-resolution mass spectrometry (LC–HRMS) analysis, all the disulfide bonds of insulin were identified without disulfide bond reshuffling. As for its metabolites, 118 were identified from rat liver S9 after incubation with insulin, including 41 with disulfide bond reshuffling. Metabolized insulin peptides with disulfide bond reshuffling were successfully verified by reduction and alkylation of the peptides along with oxidation of the disulfide bonds using meta-chloroperoxybenzoic acid (mCPBA), facilitating their fragmentation. This study provides a new strategy for the reliable and comprehensive characterization of TPPs and their metabolites with disulfide bonds.