Hydrogen deuterium exchange mass spectrometry (HDX-MS) is used to measure the exchange rate of amide hydrogens with deuterated solvent. The exchange rate of amide hydrogens is exquisitely sensitive to changes in secondary structure and solvent accessibility, and this technique has been used extensively to characterise protein folding, as well as protein-protein, protein-lipid, and protein-small molecule binding sites.

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HDX-MS experiments can be carried out with a variety of stimuli, including protein binding partners, ligands, and membrane surfaces. Importantly, HDX-MS provides information on allosteric conformational changes. Our laboratory has made significant progress in the use of HDX-MS to study membrane localised signalling complexes that have been challenging to study by other structural approaches. In tandem with this work, we have found HDX-MS as a powerful tool in the optimisation of crystallgraphic constructs, through the removal of intrinsically disordered segments. This is particularly useful in large multicomponent protein complexes, where disorder prediction software is of limited utility.

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A major advantage of our synergistic approach of using HDX-MS with X-ray crystallography is that we can combine the information gained from the high-resolution structural information of truncated constructs, with the medium resolution mass spectrometry data (HDX-MS). This allows us to look at the intact full-length proteins in our examination of the structure/function of these signalling complexes. Many of the proteins studied in our laboratory contain large intrinsically disordered regions, and it is highly unlikely for these regions to be observed in any high-resolution structural approaches. The structural mass spectrometry approach allows us to examine the role of intrinsically disordered regions in mediating interactions with other proteins, as well as in membrane binding, and protein function. Importantly this combination of techniques allows us to probe both molecular structure and protein dynamics, and this is essential to understanding how large flexible multi-domain lipid signalling complexes are regulated, both in solution, and at membrane surfaces.