HDX-MS is a versatile technique with a number of applications (Figure 3), some of which are described below.
Characterizing Protein Conformation and Comparability
HDX-MS can be used to compare different conformers of a protein to identify conformational changes. In the study of genetic disorders, HDX data can help elucidate the effects of pathogenic mutations on the protein structure, folding, and stability, offering insights into the molecular mechanisms of the diseases and providing drug design possibilities. For example, a lot of research has been conducted by HDX-MS to elucidate protein misfolding and aggregation in neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease.
Dissecting Protein-ligand Binding
HDX-MS is an indispensable tool to capture protein structural or dynamic changes induced by binding to ligands, including small molecules, peptides, nucleotides, or other proteins. The experiment typically measures HDX of the protein in both ligand-free (apo) and ligand-bound (holo) states. Differentiating the HDX profiles between the two states allows for the identification of the binding interface. Importantly, HDX-MS can reveal both orthosteric and allosteric effects on the protein molecule upon ligand binding and is sensitive enough to characterize weak binding.
Probing Chemical Modifications
Post-translational modifications (PTMs) play important roles in regulating protein functions and activities. In recent years, HDX-MS has been applied to study proteins encompassing PTMs, especially glycosylations and disulfide bonds. For example, researchers have used HDX data to characterize the glycosylation state of proteins and detect how glycosylations can impact the overall structure.
Additional applications of HDX-MS include studying conformational dynamics of membrane proteins and intrinsically disordered proteins, structural characterization of multiple-protein complexes, etc.