Structural Mass Spectrometry of Protein Assemblies

Hydrogen Deuterium Exchange coupled to MS (HDX-MS)

In theory:

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.

In Practice:

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 SynaptG2Si (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).


Structural insights into chaperone addiction of toxin-antitoxin systems.
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.
Nat Commun. 2019 Mar 7;10(1):1187.

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding.
Trcka F, Durech M, Vankova P, Chmelik J, Martinkova V, Hausner J, Kadek A, Marcoux J, Klumpler T, Vojtesek B, Muller P, Man P.
Mol Cell Proteomics. 2019 Feb;18(2):320-337.


Radu L, Schönwetter E, Braun C, Marcoux J, Kölmel W, Schmitt DR, Kuper J, Cianferani S, Egly JM, Poterszman A, Kisker C. “The intricate network between the p34 and p44 subunits is central to the activity of the transcription/DNA repair factor TFIIH” Nucleic Acids Research 45(18) : 10872-10883.

Bourgeois G, Marcoux J, Saliou JM, Cianférani S, Graille M. “Activation mode of the eukaryotic m2G10 tRNA methyltransferase Trm11 by its partner protein Trm112” Nucleic Acids Research 45(4):1971-1982.

Kadek A, Kavan D, Marcoux J, Stojko J, Felice AKG, Cianférani S, Ludwige R, Halada P, Man P. “Interdomain electron transfer in cellobiose dehydrogenase is driven by surface electrostatics” BBA General Subjects 1861(2) : 157-167.


Marcoux J, Man P, Petit-Haertlein I, Vives C, Forest E and Fieschi F. “p47phox molecular activation for assembly of the neutrophil NADPH oxidase complex” Journal of Biological Chemistry 285 (37) : 28980-28990.

Marcoux J, Thierry E, Vives C, Signor L, Fieschi F and Forest E. "Investigating Alternative Acidic Proteases for H/D Exchange Coupled to Mass Spectrometry: Plasmepsin 2 but not Plasmepsin 4 Is Active Under Quenching Conditions" Journal of the American Society for Mass Spectrometry 21 (1) : 76-79.


Marcoux J, Man P, Castellan M, Vives C, Forest E and Fieschi F. "Conformational changes in p47phox upon activation highlighted by mass spectrometry coupled to hydrogen/deuterium exchange and limited proteolysis" FEBS Letters 583(4) : 835-840.