Hydrogen/Deuterium eXchange Mass Spectrometry

Hydrogen-deuterium exchanged monitored by mass spectrometry (HDX-MS) is a powerful biophysical tool to provide information regarding protein conformation, dynamics and ligand binding.

This technique reports on the exchange of amide hydrogens on the protein backbone in the presence of deuterated solvents at a peptide level of resolution.


  • No covalent labelling required
  • No crystallization required
  • Versatile: a large range of systems can be analyzed including large biomolecular complexes, highly dynamic proteins and membrane-associated species.
  • Peptide level of resolution


Proteins are incubated in deuterated buffer causing an exchange of the labile hydrogen from the amide backbone of proteins with deuterium from the solvent.

The rate of this exchange is dependent on the solvent accessibility and the stability of the hydrogen bonding networks. It is thus strongly influenced by the folded state of the protein and its dynamic.

The changes in mass associated with the isotopic exchange is measured by collecting mass spectrometry data at several intervals after exposure to deuterated buffer.

The deuterium uptake over time reflects the conformation and dynamics under particular conditions (e.g., in the presence or absence of a ligand).

Deuterium uptake is generally measured using a ‘bottom-up’ workflow that involves quenching of the exchange reaction followed by proteolytic digestion before LC/MS-based analysis. The advantage of this approach is that it provides a degree of ‘spatial resolution’ to the data, usually in segments of around five residues on average.


  • Protein-protein interaction or protein-ligand interaction
  • Epitope mapping
  • Conformational changes


V. Vinciauskaite, G.R. Masson, Fundamentals of HDX-MS, Essays Biochem. 2023, 67, 301–314
G.R. Masson et al., Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments, Nat. Methods. 2019, 16, 595–602
D. Narang, C. Lento, D.J. Wilson, HDX-MS: An Analytical Tool to Capture Protein Motion in Action, Biomedicines. 2020, 8
R. Jia et al., Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter, Nat. Commun. 2020, 11