Investigating how intrinsically disordered regions contribute to protein function using HDX-MS

A large amount of the human proteome is composed of highly dynamic regions that do not adopt a single static conformation. These regions are defined as intrinsically disor-dered, and they are found in a third of all eukaryotic proteins. They play instrumental roles in many aspects of protein signaling, but can be challenging to characterize by biophys-ical methods. Intriguingly, many of these regions can adopt stable secondary structure upon interaction with a variety of binding partners, including proteins, lipids, and ligands. This review will discuss the application of Hydrogen-deuterium exchange mass spec-trometry (HDX-MS) as a powerful biophysical tool that is particularly well suited for struc-tural and functional characterization of intrinsically disordered regions in proteins. A focus will be on the theory of hydrogen exchange, and its practical application to identify disor-dered regions, as well as characterize how they participate in protein-protein and protein-membrane interfaces. A particular emphasis will be on how HDX-MS data can be presented specifically tailored for analysis of intrinsically disordered regions, as well as the technical aspects that are critical to consider when designing HDX-MS experiments for proteins containing intrinsically disordered regions.