| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 5135263 | Journal of Chromatography A | 2017 | 7 Pages |
â¢We combined size-exclusion chromatography, IMS, hydrogen-deuterium exchange (HDX).â¢IMS and HDX on SEC platform were used to study conformational changes in solution.â¢All three techniques were applied simultaneously to study global conformations.â¢Equilibrium-on-column conditions were used as modulated solution parameters.â¢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 (ÎHDX) 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 ÎHDX profile which showed global protein conformational changes as a difference in the number of deuterons exchanged to protons. The ÎHDX 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.
