Towards reducing back-exchange in hydrogen-deuterium exchange mass spectrometry: a feasibility study of sub-zero digestion and nano-LC separation.

In recent years, hydrogen-deuterium exchange mass spectrometry (HDX-MS) has emerged as the method of choice for characterizing increasingly complex protein assemblies. However, deuterium back-exchange remains a major limitation, as it requires rapid analysis to preserve deuterium labeling and ensure data accuracy. Reducing the temperature during the HDX-MS workflow has been recognized as an effective way to enhance deuterium retention. While sub-zero liquid chromatography (LC) has been successfully implemented, protein digestion at sub-zero temperatures has never been investigated. In this study, we evaluated the feasibility of a complete sub-zero workflow that integrates protein digestion and nano-LC, using 30% ethylene glycol (EG) in the mobile phase to prevent freezing. At - 13 °C, our nano-LC system achieved 20.5% increase in the relative retention of deuterium on peptides compared with 0 °C conditions. We also established the feasibility of performing offline digestion in the presence of EG. Moreover, we demonstrated that pepsin remains active at temperatures as low as - 19 °C, although digestion efficiency decreases under these conditions. Specifically, a 5-min digestion at - 19 °C in the presence of 30% EG resulted in an average increase of 4.7% in relative deuteration compared with a 0 °C digestion for the same duration. Overall, these results demonstrate the feasibility of sub-zero protein digestion and its integration with sub-zero nano-LC, while highlighting the trade-off between increased deuterium retention and reduced digestion efficiency. Further studies are needed to determine whether the additional deuterium retention achieved at sub-zero temperatures translates into improved HDX-MS measurements and structural interpretation.