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.

Assignment of polymorphic species of insulin analogues in ion mobility mass spectroscopy.

Electrospray ionization - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) allows the identification of protein polymorphic distribution of protein conformers and oligomers. We report the detailed identification of the species observed with commercially available pharmaceutical preparation of wild-type, regular human insulin.

Structural meta-analysis of regular human insulin in pharmaceutical formulations.

We have studied regular acting, wild-type human insulin at potency of 100 U/mL from four different pharmaceutical products directly from their final finished formulation by the combined use of mass spectrometry (MS), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), and single-crystal protein crystallography (PX). All products showed similar oligomeric assembly in solution as judged by DLS and SAXS measurements. The NMR spectra were compatible with well folded proteins, showing close conformational identity for the human insulin in the four products.

A T3R3 hexamer of the human insulin variant B28Asp.

Insulin shows a complex equilibrium between monomers and hexamers, involving varying conformers and association states. We sought to perform a structural characterization of the fast-acting human insulin variant B28Asp ("aspart"). Small-angle X-ray scattering measurements reveal similar globular behavior in both the aspart and regular human insulin, with a Rg of 19Å and a Dmax of approximately 50Å, indicating similar mean quaternary assembly distribution.