HDXperts
Hydrogen Deuterium Exchange Mass Spectrometry Services
What is Hydrogen Deuterium Exchange Mass Spectrometry ?
In polypeptides, all the hydrogens bounded to heteroatoms (e.g. –OH, -NH, -SH, COOH, –CONH2, –NH2) are constantly exchanged for other hydrogens present in the surrounding aqueous solvent. In this context, polypeptides can easily incorporate deuterium if exposed to heavy water (deuterated water).
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This reaction – called hydrogen/deuterium exchange, HDX - is highly correlated to the chemical nature of the heteroatom and its intra- and inter-molecular interactions.
An additionally characteristic is that although the incorporation of deuterium does not have any practical effects on proteins, the presence of a neutron in the hydrogen nucleus can be detected by NMR or mass spectrometry. Therefore, the HDX reaction can be utilized as an innocuous labeling marker to monitor protein structure.
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Now, for most hydrogens, the HDX exchange is short-lived, and as soon as the polypeptide chain is in contact with a water-containing solution, the exchanged deuterons are again replaced by hydrogen.
This phenomenon is called “back exchange”, and it is very fast for deuterons bounded to heteroatoms located at the side chain of the amino acid residues. Fortunately, under acidic conditions (pH ~ 2.3) and near zero °C temperatures, the back exchange at the peptide backbone amide group slows to time windows compatible with analysis by liquid chromatography and mass spectrometry (LC-MS).
In this manner, the degree of deuterium incorporation into the hydrogen amide group of the peptide bond can be monitored by MS since each D atom is one mass unit heavier than an H atom.
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The possibility to probe the kinetics the hydrogen exchange rate at the amide group of polypeptide chains provides a unique window to probe protein structural dynamics, as well as the characterization of binding interfaces. The later is achieved by comparing the local amide NH kinetics of a protein in the presence and absent of a ligand (small molecule or another polypeptide).
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