Polymorphic distribution of proteins in solution by mass spectrometry: The analysis of insulin analogues

The characterization of conformational and oligomeric distribution of proteins is of paramount importance for the understanding of the correlation between structure and function. Among the bioanalytical approaches currently available, the electrospray ionization-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry (IMS) is the best suited for high resolution identification with high sensitivity, allowing the in situ separation of oligomeric and conformational species. We tested the performance of the ESI-MS technique along with the IMS separation approach on a broad variety of insulin and insulin analogues with distinct oligomeric distribution pattern. The measurement of commercial insulin allowed the identification of species ranging from monomers to hexamers and their complexes with zinc ions. Dissimilar distribution profile for regular insulin as a function of formulation component and among the insulin analogues were observed by ESI-IMS-MS but not by ESI-MS along, crystallographic assays or size-exclusion chromatography. These data suggest the additional suitability of ESI-IMS-MS in conformational and oligomeric profiling of biomacromolecules and biopharmaceuticals. The easiness of the technique provides further motivation for its application in the characterization of both raw and formulated protein biopharmaceuticals in routine and comparability exercises. •Proteins can populate a variety of conformational and oligomeric states.•CD, SEC, MALDI-ToF and crystallography display limited access to equilibrium conformational diversity.•Ion mobility spectrometry (IMS) evidenced dissimilar conformational and oligomeric distribution of insulin.•IMS showed sensitive to variables such as solution composition and sequence variants.•IMS-MS may be considered a tool in profiling the oligomeric and conformational plasticity of biopharmaceuticals.