Comprehensive Internal Standards for Hydrogen-Deuterium Exchange Mass Spectrometry

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a widespread tool for studying protein structure and dynamics, but data comparability across different experimental conditions, instrument platforms, and laboratories remains challenging. Here, we design, synthesize, and validate a novel set of imidazolium-based internal exchange reporters (IERs) that readily integrate with HDX-MS to provide a direct measure of exchange conditions. The set of eight IERs are designed to have a single discrete exchangeable site to sample conditions over exceptionally broad time scales, from milliseconds to days, while also exhibiting desirable chromatographic behavior. We demonstrated the utility of these IERs and the associated computational tools through a series of comparative HDX-MS studies involving well-studied proteins where reaction conditions like pH and deuterium content were offset. Through unambiguous referencing of exchange reaction conditions, this novel set of internal standards enables rigorous cross-experiment comparisons and will improve HDX-MS-derived models of protein dynamics.