Empirical Method To Accurately Determine Peptide-Averaged Protection Factors from Hydrogen Exchange MS Data

Amide hydrogen exchange experiments measured by mass spectrometry have become commonplace to study protein structural dynamics; however, the underdetermined nature of these measurements render extraction of exchange rates unreliable at the level of individual peptides. This prevents orthogonal verification of results and severely limits interpretation of the data. This work describes an easy-to-implement empirical method to determine the change in an observed rate constant or the average change in multiple rate constants as compared to some reference condition. This allows direct empirical computation of the average protection factor (PF) for peptides in isolation requiring no knowledge of actual rate constants themselves. Benchmarking the method by comparison of average peptide PFs with site-resolved NMR-derived PFs demonstrates high reliability and accuracy. This empirical method provides the first universally reliable strategy for recovering subglobal structural physics from individual peptides and, in doing so, standardizes the hydrogen exchange experiments measured by bottom-up mass spectrometry (HX MS), simplifies interpretation, and facilitates clear communication of the results.