Mice are the most common model animal for the study of diabetes and insulin. Mice possess an insulin two-gene system encoding mouse insulin 1 (Ins1) and insulin 2 (Ins2). Ins2 is the orthologue to human insulin. Our work has established a workflow utilizing liquid chromatography coupled to ion mobility spectrometry-mass spectrometry (LC-IMS-MS) to distinguish mature Ins1 and Ins2 from isolated mouse islets. We hypothesize the structural differences between the two proteins may play a role in stability and aggregation.

We focus our studies on the B-chains of Ins1 and Ins2, as they differ only by two residues (P9S and K29M) in the B-chain. The B-chains contain an amyloidogenic region which is thought to result in cytotoxic aggregation and deactivated insulin. The P9S sequence difference may modulate insulin oligomerization and stability in physiological environments.

We have monitored non-enzymatic peptide cleavages which provide information on protein structure. Generally, peptide bonds within a folded, protected region are less likely to be cleaved than those from unstructured, flexible regions. This aspect, in addition to non-native disulfide bond formation, was investigated with LC-IMS-MS. The end goal is to correlate these findings with the structural differences observed in mature Ins1 and Ins2, of which we found that Ins1 is more folded (structurally more compact) than Ins2.

Our work rationalizes the increased degradation of Ins2 through comparisons of non-enzymatic bond cleavages. The sites of fragmentation for the Ins1 B-chain are in the middle region, which is protected by the A-chain in the mature protein. Conversely, the B-chain of Ins2 is more prone to be truncated at the C-terminus which is not well protected by the A-chain in the mature protein. Consequently, the mature Ins2 protein experiences higher degree of truncation, consistent with our observation from isolated mouse islets that Ins2 is less stable than Ins1.