30 demonstrated how HDX-MS can provide mechanistic and dynamic detail to cryo-EM structures, and how HDX-MS can aid modeling of X-ray structures within the cryo-EM density. 29 revealed a new allosteric mechanism for interrupting the antiapoptotic binding of MCL-1 to BH3 domains, providing a new avenue for cancer therapy. 28 localized the substrate-dependent partial unwinding of transmembrane helices, which facilities substrate translocation using HDX-MS. 27 discerned the basis of isotype specificity of pharmaceutical compounds by determining single-residue exchange rates. 26 determined how an increase in temperature alters the Dengue virus capsid structure, resulting in an alteration in antibody-binding mode. 25 demonstrated how HDX-MS can be used in combination with X-ray crystallography and cryo-electron microscopy (cryo-EM) when determining the mechanism of exonuclease activity of DnaE1. 24 localized dehydration and zinc-activated disorder-to-order transitions in abiotic plant stress response proteins using HDX-MS. It is not surprising that the number and diversity of studies that use some variant of the HDX-MS method is increasing year over year 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 (Fig. A reduced barrier to entry has accompanied these developments, resulting in an explosion in the amount of HDX-MS data being generated. The analysis of large multi-subunit proteins no longer takes weeks or months.
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Data analysis software has progressed markedly 20, 21, 22, increasing processing speeds by several orders of magnitude over the past ten years. Additional advances in liquid chromatography (LC)-MS technology and automation have greatly increased the user base for HDX-MS. Mass spectrometry has been the method of choice for detecting HDX since the 1990s 17, 18, because it can accommodate large proteins (>100 kDa), accept low concentrations (less than micromolar) and tolerate complex sample matrices 19. Measurement of protein HDX dates back to the 1950s 14, 15, with much of the work in the 1970–1980s using NMR spectroscopy as the method of detection 16. Of particular relevance in industry, HDX-MS also excels at epitope mapping and the characterization of biotherapeutics 12, 13. HDX-MS is a very versatile technique 5 and can be used to examine conformations of individual proteins or large protein complexes 6, locate protein sites directly or indirectly involved in binding 7, probe for allosteric effects 8, monitor the folding dynamics of protein domains 9, examine intrinsic disorder 10 and provide insights into protein–membrane interactions 11. The rate of this exchange is dependent on the folded state of the protein and its dynamics (particularly the stability of hydrogen bonding networks) and the intrinsic chemical properties of the underlying amino acid sequence 2, 3, 4. HDX-MS measures changes in mass associated with the isotopic exchange between amide hydrogens of the protein backbone and its surrounding solvent. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful technique that can provide insights into protein behavior by serving as a link between structure, conformational dynamics and function 1. Nature Methods volume 16, pages 595–602 ( 2019) Cite this article Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments