Abstract: Hydrogen-deuterium exchange nuclear magnetic resonance (HDX-NMR) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) are important tools for studying protein structure and dynamics. In recent years, they have been widely applied in elucidating protein conformational changes. HDX-NMR, by directly detecting NMR signals post hydrogen-deuterium exchange, offers dynamic information about proteins at the single amino acid resolution, making it particularly suitable for investigating slowly exchanging regions and conformational changes over extended time scales. Conversely, HDX-MS combines the advantages of hydrogen-deuterium exchange with high-resolution mass spectrometric analysis, allowing for the determination of the solution-state structure of proteins under near-physiological conditions and proving effective for complex systems such as macromolecular complexes and membrane proteins. Both techniques boast unique advantages compared to traditional structural biology methods (e.g., X-ray crystallography and cryo-electron microscopy). Notably, HDX-MS is characterized by its high sensitivity and minimal sample requirements, while HDX-NMR excels in site resolution and kinetic analysis. Recently, enhancements in data processing methodologies and experimental techniques for HDX-MS and HDX-NMR have broadened their application prospects in areas such as protein conformational changes, drug screening, and protein folding. This review outlines the fundamental principles, experimental protocols, and data analysis methods associated with HDX-MS and HDX-NMR, systematically comparing their similarities and differences in protein research. Furthermore, we discuss the complementary nature of HDX-MS and HDX-NMR, as well as their integration with other structural biology techniques, and provide insights into potential future directions for development, aiming to offer valuable references for related research.

Key words: HDX-NMR, HDX-MS, Protein, Structural Dynamics.