Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation.

Hydrogen Deuterium Exchange (HDX) Mass Spectrometry (MS) is a sensitive analytical technique that provides information on protein conformation and dynamics in solution. It is commonly used in the study of protein-ligand and protein-protein interactions and more recently in the pharmaceutical industry for epitope mapping, screening drug candidates and in the comparison of biopharmaceuticals to biosimilars. HDX-MS monitors the exchange of protein backbone hydrogen atoms with deuterium in solution. Recent advancements in HDX automation and data analysis, have taken the emphasis off developing a fundamental understanding of HDX, which is still lacking. This tutorial review will cover the different mechanisms of exchange and how the exchange reaction is affected by various factors. We also explore the basis of data analysis and the difficulties that often arise in the interpretation of site-specific and segment-averaged HDX data, such as overlapping isotopic distributions and correct identification of peptides. Initial data analysis generates a list of peptides and the deuterium incorporation of each peptide at each labeling time point, i.e., a set of deuterium uptake profiles. Data interpretation and error analysis is subsequently required to ensure that deuterium uptake profiles accurately reflect conformational dynamics in solution. Finally, this review will also discuss the different ways in which HDX data can be represented and how the data can be interpreted.