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, protein-protein interfaces, and small molecule binding sites.
HDX-MS experiments are carried out by exposing a protein of interest to deuterated solvent for a set period of time, followed by quenching in a low pH buffer where exchange rates are dramatically decreased, followed by fragmentation of the protein either by immobilised proteases or within a mass spectrometer. The mass of these fragments can be determined within a mass spectrometer, and deuterium incorporation can be determined through examining the change in the mass centroid.
HDX-MS experiments can be carried out with a variety of stimuli, including protein binding partners, ligands, and membrane surfaces. Defining the difference in exchange rates between these conditions allows for the mapping of protein binding interfaces, however, importantly HDX-MS also provides information on allosteric conformational changes. Another of the main uses of HDX-MS is for the determination of disordered regions within proteins as an aid for the design of optimised crystallography constructs. The removal of intrinsically disordered regions has been shown to dramatically increase the probability of crystallisation. This is particularly useful in large multicomponent protein complexes, where disorder prediction software is of limited utility.
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.