Hydrogen-deuterium exchange enables to detect conformational differences between two protein samples.

The proteins or protein complexes are diluted in a deuterated buffer and hydrogen atoms from the peptide bonds are exchanged with the deuterium atoms of the solution. The rate of exchange of each amide hydrogen depends on its solvent accessibility and involvement in the stabilization of secondary structures. By determining the rate of deuterium exchange, we can identify which regions are solvent-accessible and which ones are hidden in the core of the protein.

This expanding method can thus be used to study protein dynamics, compare protein conformations (in different buffer conditions, after mutation), or to identify protein-protein or protein-ligand interfaces.

Proteins (~ 5 µl at 0.5-10 µM) are diluted 20 times in a deuterated buffer (at physiological pH) before a rapid quench at 4°C and pH 2.6.The quenched solution is then digested online (pepsin column) and peptides are desalted on a C18 cartridge before being separated on a C18 column at 4°C. The whole sample preparation, digestion and MS analysis is automated thanks to our Automated HDX robot (LeapTec) coupled to the Synapt G2-Si (Waters) (Figure 1). The data analysis is also entirely automated (Figure 2). Figure 1. Automated HDX-MS solution including the sample preparation robot (LeapTec), the M-Class Acquity UPLC, the HDX-module for protein digestion and peptide separation at 4°C and the SynaptG2Si (Waters). Figure 2. Dynamix (Waters) software automatically generates sequence coverage maps (A), extracts the mass increment of each peptide at each timepoint (B) to draw deuteration curve (C). Heatmaps are then generated (D) and can be imported to Pymol, if a structure of the protein of interest is available (E).


Lesne J, Locard-Paulet M, Parra J, Zivković D, Menneteau T, Bousquet MP, Burlet-Schiltz O, Marcoux.Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependant inter-ring crosstalks. Nature Communications 11(1):6140.



Guillet V, Bordes P, Bon C, Marcoux J, Gervais V, Sala AJ, Dos Reis S, Slama N, Mares-Mejía I, Cirinesi AM, Maveyraud L, Genevaux P, Mourey L. Structural insights into chaperone addiction of toxin-antitoxin systems. Nature Communications 10(1):782.


Bouyssié D, Lesne J, Locard-Paulet M, Albigot R, Schiltz O, Marcoux J. HDX-Viewer: interactive 3D visualization of Hydrogen-Deuterium eXchange data. BioInformatics 35(24):5331-5333.