Developments in hydrogen/deuterium exchange mass spectrometry at sub-zero temperatures

HDX-MS has become a popular and established method for studying the dynamic conformational landscape and interactions of proteins in solution. Over the years, HDX-MS has been used to analyze increasingly complex protein systems in terms of size, purity, and biologically relevant conditions. For example, HDX-MS has been employed to study membrane proteins in their native lipid membranes from bacteria, intact viruses, and individual proteins within living cells. However, these exciting and complex new applications pose significant challenges to traditional HDX-MS experiments, particularly the need to perform sample pretreatment and LC separation on a short timescale at zero degrees to prevent inadvertent deuterium labeling information loss (back-exchange). A promising development in HDX-MS is the use of sub-zero temperature separation to reduce back-exchange and enable extended chromatographic separation. In this review, we aim to provide an overview of the current state of sub-zero temperature HDX-MS and the challenges associated with its use. • HDX-MS is a powerful method for analyzing conformational dynamics of proteins. • Sub-zero temperature HDX-MS decreases the back-exchange that can otherwise limit applications of HDX-MS. • Improved back-exchange of HDX-MS workflows can improve the data quality and sensitivity of the technique and enable analysis of increasingly complex protein systems.