Hydrogen–Deuterium Exchange

H/D exchange experiments are based on the chemical reaction of replacing covalently bonded hydrogens with deuterium atoms to reveal the tertiary structure information of proteins.

Because H/D exchange depends on the solvent accessibility/hydrogen bonding of the amide hydrogen, H/D exchange as an analytical technique provides a good probe of protein conformational dynamics and interactions.

HD exchange

As depicted schematically above, when a protein is diluted 20-fold in D2O buffer, labile hydrogen atoms on the molecule will exchange with deuterium. Labile hydrogens, such as those on primary amines and on the side chains, exchange nearly instantaneously. Backbone amide hydrogens exchange at rates that depend on the local backbone conformation and dynamics. In highly dynamic unstructured regions, the exchange reaction proceeds in milliseconds to seconds, whereas hydrogen-bonded amide hydrogens exchange much slower (minutes to days). The rate of deuterium incorporation is characteristic of local structure and dynamics.

quench digest

H/D exchange coupled with mass spectrometry measures the deuteration rate. H/D exchange experiments with different reaction periods are quenched by lowering pH to ~2.5. Quenching is followed by proteolysis, chromatographic separation, and mass analysis. The quenching locks the pattern of deuteration imprinted on amide backbone before HPLC and MS. The mass shifts that arise through the incorporation of deuterium into the protein are monitored by mass spectrometry, and localized backbone structure and dynamics can be determined.

Differences in H/D exchange for free and complexed protein serve to map the contact surfaces in the complex (based on protection against H/D exchange on binding of ligand(s) or other protein(s)). Conformational changes due to point mutations can also be charcterized. Compared to x-ray diffraction or nuclear magnetic resonance, H/D exchange is not limited by the protein size or quality of crystals, and proteins can be analyzed in a buffer system closer to physiological conditions. Ultrahigh mass resolution enables resolution of dozens of peptides, and is essential for extending the technique to large proteins and complexes.

This technique can be used with the following instruments:

Related Publications

S. Kazazic, et al, Automated Data Reduction for Hydrogen/Deuterium Exchange Experiments, Enabled by High-Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Journal of the American Society for Mass Spectrometry 21(4), 550-558 (2010) Read online 

H. M. Zhang, et al, Enhanced Digestion Efficiency, Peptide Ionization Efficiency, and Sequence Resolution for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Analytical Chemistry 80(23), 9034-9041 (2008) Read online 

H. He, et al, Method for Lipidomic Analysis: p53 Expression Modulation of Sulfatide, Ganglioside, and Phospholipid Composition of U87 MG Glioblastoma Cells, Analytical Chemistry 79(22), 8423-8430 (2007) Read online 

M. J. Chalmers, et al, Probing Protein Ligand Interactions by Automated Hydrogen/Deuterium Exchange Mass Spectrometry, Analytical Chemistry 78(4), 1005-1014 (2006) Read online 

For more information please contact Amy McKenna, Manager, ICR User Program.

Last modified on 16 November 2015