Mass Spectrometric Characterization of Native Insulin Hexamers and Non-Native Heptamers: Formation and Stability...

Insulin aggregation and oligomerization present significant challenges in both therapeutic formulation and fundamental studies of amyloid formation, particularly due to the transient and heterogeneous nature of early-stage oligomers. Here, we employ ion mobility-mass spectrometry (IM-MS) to characterize the oligomeric distributions of human insulin and two clinically relevant analogs, aspart (rapid-acting) and glargine (long-acting), in excipient-containing solutions designed to stabilize native forms of insulin and under aggregation-inducing conditions. Comparison of insulin analogs revealed distinct aggregation propensities that correlate with each analog's therapeutic design. In general, stable zinc-coordinated hexamers formed most readily in the presence of excipients, and various oligomers formed under aggregation-inducing conditions, with a noticeable prevalence of heptamer formation. We further investigated the structure and stability of native hexamers and non-native heptamers using collision-induced dissociation and collision-induced unfolding experiments. Although the expected single-monomer ejection was the main dissociation pathway for both species, zinc-coordinated hexamers also dissociated into two zinc-adducted trimers, whereas heptamers dissociated into dimer/pentamer and trimer/tetramer pairs. Gas-phase unfolding indicated conservation of subunit tertiary structure in hexamers and no distinct folded structures in heptamer subunits. Overall, this work demonstrates the utility of IM-MS as a rapid, high-resolution platform for probing insulin aggregation pathways and evaluating current and future insulin analog formulations.