Hydrogen Deuterium Exchange Mass Spectrometry Service

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and important tool used to study protein conformation and dynamics, protein-protein interactions, protein-small molecule interactions, and protein-RNA interactions. HDX-MS takes advantage of the naturally proton exchange occurring at amides on proteins, and deuterium can replace the protein backbone hydrogens upon exposure of the protein to a D2O-based buffer. According to the protein's structure, the exchange rates varies. If the hydrogen-bonded segments are tight, the exchange rate is low; the exchange rate is high when the regions are disordered. The hydrogens on the protein surface will be exchanged with deuterium atoms after immersing the protein or protein complex into the deuterium solution. The exchange will be then detected and quantified by mass spectrometry since the deuterium is heavier than hydrogen. HDX-MS can be utilized to detect single protein's and protein complexes' structure and conformation information, as well as protein-protein or protein-ligand interaction sites and conformational changes induced by posttranslational modifications (PTMs) in protein complexes.

Since the high order structure and conformational dynamics of proteins are closely related to the efficacy of biopharmaceutical agents, it is essential to well acknowledge protein higher order structure, conformation, and interactions during drug discovery and development. Creative Proteomics provides a precisive HDX-MS service to discover information on protein conformation, dynamics, interaction sites in various types of samples, including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), viral capsids, and so on. Our professional scientists will optimize procedures to fulfill the requirements during all stages of drug discovery and development.

Hydrogen Deuterium Exchange Mass Spectrometry Service

We Can Provide but Not Limited to:

  • Identification of protein conformation
  • Epitope mapping
  • Determination of protein-protein interaction, protein-small molecule interactions, and protein-RNA interactions
  • Assessments of dynamical comparability
  • Evaluation of structural modification

Technology Platform:

In HDX experiments, protein will be first labeled. Two main approaches can be performed to label proteins by HDX: continuous labeling and pulsed labeling. In continuous labeling approach, several aliquots of the protein are immersed in the deuterated buffer under conditions that keep the native conformation of the protein. Each aliquot of the protein sample is incubated in the buffer for a certain period time and then labeling is ended. This approach can monitor the deuterium exchange as a function of time, and be used to investigate conformational dynamics of a protein under equilibrium conditions. In pulsed labeling approach, a protein is first perturbed by chemical denaturant, pH change, temperature change, etc. Then the protein is immersed in deuterated buffer with higher pH for a short time. This approach allows HDX occur only at positions that are solvent-exposed but not involved in hydrogen bonds. In order to reduce the exchange rate and make an end to the reaction during analysis, the deuterated protein will be placed in a low pH buffer at low temperature following labeling. Subsequently, MS will be performed to measure the deuterium incorporation into the proteins.

Hydrogen Deuterium Exchange Mass Spectrometry Service

Advantages of HDX-MS

  • High sensitivity and accuracy: precise characterization of proteins with different molecular weight from ~10 kDa to ~2MDa.
  • Good batch to batch reproducibility.
  • Advanced system: Orbitrap Exploris™ 240 Mass Spectrometer, etc.
  • Rapid turnaround time: 1 to 8 weeks to provide comprehensive report depending on sample size and service chosen.
  • Customized service: Optimized methods will be customized based on your sample type, size and service.

Creative Proteomics's experienced scientists can provide extensive analysis of protein by HDX-MS, including conformation, interaction, etc. Clear, comprehensive written reports, recommendations and protocols, and customized services will be provided to help customers solve analytical and technical problems.


  • Hudgens J.W., Gallagher E.S., Karageorgos I., et al., Interlaboratory Comparison of Hydrogen−Deuterium Exchange Mass Spectrometry Measurements of the Fab Fragment of NISTmAb. Anal. Chem. 2019, 91, 7336−7345.
  • Deng B., Lento C., and Wilson D.J., Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development e A review. Analytica Chimica Acta 940 (2016) 8e20.
  • Wei H., Mo J., Tao L., et al., Hydrogen/Deuterium Exchange Mass Spectrometry for Probing Higher Order Structure of Protein Therapeutics: Methodology and Applications. Drug Discov Today. 2014 January; 19(1): 95–102.

*For Research Use Only. Not for use in the treatment or diagnosis of disease.

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