Principle and Procedure of Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS)

Hydrogen deuterium exchange mass spectrometry (HDX-MS) can be used to study protein spatial conformation, protein structure dynamics, protein-interaction sites, and to identify protein surface active sites.

Various applications of HDX-MS techniqueVarious applications of HDX-MS technique (Narang et al., 2020)

Principle of HDX-MS

When a protein is diluted with a heavy water solution, unstable hydrogen atoms in the protein molecule are exchanged with deuterium atoms in the heavy water solution. Unstable hydrogen atoms mainly include those in the protein molecule that are attached to nitrogen, oxygen and sulfur in the branched chain, as well as those in the amino group in the amide bond of the main chain. Those hydrogen atoms attached to carbon atoms do not exchange hydrogen for deuterium. The rate of hydrogen-deuterium exchange of unstable hydrogen atoms located on the branched chain is very fast. The exchanged deuterium atoms are quickly exchanged down by the hydrogen atoms in solution after leaving the high concentration of heavy water in the later stages of the experiment, and are difficult to detect by mass spectrometry. In contrast, the rate of hydrogen-deuterium exchange of the main chain amino hydrogen atoms is relatively slow and can be detected by mass spectrometry. Therefore, the exchange process of the main chain amino hydrogen atom with the deuterium atom in heavy water solution can be used to study the protein structure.

Principle of HDX-MS

The rate of hydrogen-deuterium exchange of the main chain amino hydrogen atoms is most closely related to the structure of the protein at a given reaction pH and temperature. When the structure of proteins is changed by external influences (e.g., changes in the degree of contact between the protein and the solution or in the number of hydrogen bonds in the protein molecule), their level of hydrogen-deuterium exchange changes accordingly. Deuterium atoms have a molecular weight one Dalton greater than hydrogen atoms, and deuterated proteins have a greater mass. The mass spectrometry analysis of deuterated proteins after enzymatic digestion can determine the rate of hydrogen-deuterium exchange of different sequence fragments of the protein and thus derive information on the spatial structure of the protein.

HDX-MS Experimental Procedure

The most widely used HDX-MS experiments are currently based on a bottom-up analysis strategy. The process includes in-solution deuterium labeling, quenching of the labeling reaction, proteolytic desalting, peptide separation by UHPLC system, mass spectrometry detection, and data analysis.

1) Hydrogen-deuterium exchange reaction of the sample

The prepared aqueous protein solution is diluted 15-20 times with heavy water for the hydrogen-deuterium exchange reaction. A high concentration of heavy water (more than 90%) ensures that the reaction is dominated by the exchange of hydrogen atoms to deuterium atoms.

2) Termination of the reaction

The reaction is terminated at different time points (0, 10, 30, 60, 300, 900 and 10 000 s) by lowering the pH of the sample solution to 2.5 while the temperature is lowered to 0 °C. Due to the large number of samples that cannot be post-treated in time, samples need to be stored at ultra-low temperatures, e.g. with liquid nitrogen.

3) Sample enzymatic digestion

Enzymatic digestion with immobilized protease under termination conditions (generally controlled between 5 and 10 min) is performed to obtain a large number of deuterated peptides. If the protein molecule contains disulfide bonds, the enzymatic digestion must be accompanied by the addition of tricarboxymethyl phosphate, which is capable of reducing the disulfide bonds under acidic conditions.

4) Mass spectrometry detection

Mass spectrometric detection is the most critical step in the HDX-MS process. The commonly used mass spectrometry include high performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). 

When using LC-ESI-MS, the deuterated peptides are separated in liquid phase at low temperature and then immediately detected by mass spectrometry. For MALDI-TOF MS, the deuterated peptides are mixed with the matrix at low temperature and co-crystallized under moderate vacuum before being detected by mass spectrometry. Undeuterated enzymatic peptides need to be pre-identified by MS/MS.

5) Data analysis

As the exchange time increases, the peptides undergoing the exchange reaction become larger due to their mass. The mass spectral signal will gradually move to the direction of high mass-to-charge ratio (m/z), and the change curve of exchange rate of different peptides in different hydrogen-deuterium exchange reaction time is obtained. By comparing the hydrogen-deuterium exchange curves of the same peptide in different states (before and after protein folding, before and after formation of multimers or complexes, before and after binding to ligands, etc.), information on the spatial structure or structural dynamics of the protein can be obtained.

Schematic overview of a conventional bottom-up/local HDX-MS workflowSchematic overview of a conventional “bottom-up/local” HDX-MS workflow (Trabjerg et al., 2018)

Advantages of HDX-MS

1) Protein is in its natural state in solution

2) Dynamic protein change process can be analyzed

3) High sensitivity of mass spectrometry detection, low amount of protein samples (microgram level)

4)High speed of mass spectrometry analysis, which greatly shortens the experimental cycle

5) Mass spectrometry equipment is very common, reducing the threshold of experiments


  1. Narang, D., Lento, C., & J. Wilson, D. (2020). HDX-MS: an analytical tool to capture protein motion in action. Biomedicines, 8(7), 224.
  2. Trabjerg, E., Nazari, Z. E., & Rand, K. D. (2018). Conformational analysis of complex protein states by hydrogen/deuterium exchange mass spectrometry (HDX-MS): Challenges and emerging solutions. TrAC Trends in Analytical Chemistry, 106, 125-138.
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