Hydrogen deuterium exchange mass spectrometry (HDX MS)

Powerful tool for protein tertiary structrual analysis

We separate this technique from the rest of protein characterization methods because of its unique capability for tertiary structural characterization. In this method, protein of interest is incubated in deuterated water to allow exchange of hydrogens on the backbone amides with deuterons in the solvent. The exchange rate is very sensitive to changes in solvent accessibility and structure flexibility. Therefore, HDX MS detects structural alteration due to ligand binding, protein-protein interaction and protein modifications. For example, the epitope region on the antigen targeted by monoclonal antibody will display reduced exchange rate in the presence of antibody relative to antigen alone and can be identified by HDX MS. Following the exchange, the reaction is quenched with acidic pH and low temperature. The proteins are digested with pepsin or other acidic proteases and analyzed on a Q-TOF MS. The chromatography system is maintained at low temperature to minimize back exchange. The deuterium incorporation is calculated from the mass increase of the peptide ions. HDX MS has a variety of important applications including epitope mapping of monoclonal antibodies, ligand binding identification, structural biology and biosimilar comparability study. Comparing to X-ray crystallography, HDX MS requires much less material, is much faster and can tolerate some degree of impurity or heterogeneity in the sample. The key technical challenge in HDX MS is to achieve good sequence coverage during the short digestion time. Our proprietary digestion method allows increased sequence coverage of complex proteins such as glycan and disulfide bond rich proteins which are resistant to the common digestion protocol in the literature. NovaBioAssays is currently the only company providing HDX MS service.