Systematic Evaluation of the Impact of Quench Conditions on Deuterium Back-Exchange in HDX-MS

Abstract Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is widely used to characterize protein structure and dynamics, but accurate measurements depend on minimizing deuterium back-exchange during sample preparation. Although quench buffers typically contain denaturants and reducing agents under low pH conditions, the effects of quench solution composition and quench duration on deuterium preservation have not been systematically investigated quantitatively. Here, we quantitatively evaluated the influence of denaturant concentration, reducing agent concentration, quench pH, and quench duration on deuterium back-exchange using deuterated phosphorylase b protein and bovine serum albumin (BSA) digest peptides. Variations in denaturant and reducing agent concentrations produced only negligible changes in deuterium retention, whereas increasing the quench pH from 2.5 to 4.0 resulted in an approximately 4 percentage point decrease in average percent deuterium, identifying high quench pH as the condition exerting the greatest influence on deuterium back-exchange among the conditions investigated. Increasing quench duration from 1 to 10 min reduced average deuterium content by approximately 6 percentage points for BSA digest but only 1.6 percentage points for phosphorylase b, highlighting the protective effect of residual protein structure during the quench step. These findings provide practical guidance for selecting HDX-MS quench conditions that balance deuterium preservation with efficient protein processing while improving the accuracy and reproducibility of HDX-MS measurements.