Direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques provide an increasingly popular route to determine quantitative information on protein-protein and protein-ligand interactions. When combined with hydrogen deuterium exchange (HDX), details on protein stability and complex conformation can be obtained; however, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting gas phase results. Zhu et al.  and Powell and Fitzgerald  have outlined LC-MS based techniques to probe the solution phase properties of the protein-ligand system in question. We here have taken the well characterised soluble immunophilin protein cyclophilin A, and examined it in complex with its endogenous ligand cyclosporin A. This ligand is widely used as an immunosuppressant following organ transplant, and the complex provides a basis for drug discovery efforts. We have used direct infusion, coupled with HDX, gas phase HDX and also the LC-HDX techniques PLIMSTEX and SUPREX. Results from each of these four HDX methodologies are presented here and discussed critically. From our direct infusion we find that there are 2 observable hydrogen populations in the protein, a very fast exchanging population, and a slower group. The exchange rate of both is lowered in the presence of the ligand. For PLIMSTEX we find a Kd for ligand binding of 321±128nM, which is within one order of magnitude of values previously reported. SUPREX under a variety of conditions provides a range of Kd values, but when we average these for experimental error we obtain a Kd of 7.11±0.29nM which agrees well with measurements from other studies including via SUPREX. Finally gas phase HDX of the native complex shows more than 3 distinct populations of exchangeable hydrogens, for both the apo- and the holo protein consistent with an unfolding and refolding of the protein in the gas phase. The different techniques are compared with respect to the advantages and disadvantages they bring to the study of this protein-ligand system. © 2010 Elsevier B.V.
|Number of pages||8|
|Journal||Journal of Chromatography A|
|Publication status||Published - 22 Oct 2010|
- Cyclophilin A
- Gas phase HDX