Semi-automated screen for global protein conformational changes in solution by ion mobility spectrometry-massspectrometry combined with size-exclusion chromatography and differential hydrogen-deuterium exchange

- We combined size-exclusion chromatography, IMS, hydrogen-deuterium exchange (HDX).
- IMS and HDX on SEC platform were used to study conformational changes in solution.
- All three techniques were applied simultaneously to study global conformations.
- Equilibrium-on-column conditions were used as modulated solution parameters.
- A semi-automated experimental setup based on the use of SEC was applied.

Development of methodologies for studying protein higher-order structure in solution helps to establish a better understanding of the intrinsic link between protein conformational structure and biological function and activity. The goal of this study was to demonstrate a simultaneous screening approach for global protein conformational changes in solution through the combination of ion mobility spectrometry-mass spectrometry (IMS-MS) with differential hydrogen-deuterium exchange (ΔHDX) on the size-exclusion chromatography (SEC) platform in a single on-line workflow. A semi-automated experimental setup based on the use of SEC on-column conditions allowed for tracking of protein conformational changes in solution as a function of acetonitrile concentration. In this setup, the SEC protein elution data was complemented by the ΔHDX profile which showed global protein conformational changes as a difference in the number of deuterons exchanged to protons. The ΔHDX data, in turn, was complemented by the changes in the drift time by IMS-MS. All three orthogonal techniques were applied for studying global higher-order structure of the proteins ubiquitin, cytochrome c and myoglobin, in solution simultaneously. The described approach allows for the use of a crude sample (or mixture of proteins) and could be suitable for rapid comparison of protein batch-to-batch higher-order structure or for optimizing conditions for enzymatic reactions.