Raman spectroscopy monitoring of hydrogen-deuterium exchange kinetics in lyophilized lysozyme at controlled humidity

Abstract

Hydrogen-deuterium exchange (HDX) is a well-established technique for probing protein structure, dynamics, and solvent accessibility. Traditionally, HDX is combined with mass spectrometry, which requires quenching the exchange reaction at discrete time points, followed by proteolytic digestion and chromatographic separation. This approach is labor-intensive, time-consuming, and does not allow continuous monitoring of the exchange process on a single sample. In this work, we propose an approach based on Raman spectroscopy, which enables real-time, non-destructive monitoring of HDX kinetics directly from the same sample throughout the entire exchange process. The exchange is performed in the gas phase by exposing lyophilized protein samples to D2O vapor at controlled relative humidity. The exchange process was monitored via the intensity changes of vibrational bands corresponding to NH/OH (3200–3700 cm⁻¹) and ND/OD (2050–2700 cm⁻¹) stretching modes, normalized to the CH stretching band near 2930 cm⁻¹, which remains unchanged during the exchange. Lyophilized lysozyme samples were exposed to D2O vapor at two different relative humidities 98% and 11%. The kinetics of deuteration were recorded over time, revealing a rapid exponential increase in the ND/OD-to-CH intensity ratio, reaching saturation after approximately 3.5 hours for both humidity levels. The characteristic relaxation times obtained from stretched exponential fits were similar for both conditions approximately 86 minutes, suggesting that the deuteration rate in lyophilized powder is largely independent of water vapor pressure within the studied range. This work was supported by Russian Science Foundation (Grant No. 25-12-00083).

Speaker

Dobrynina Elizaveta Aleksandrovna
Institute of Automation and Electrometry, Russian Academy of Sciences, Novosibirsk
Russia

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