Analysis of Quaternary Structure by Hydrogen Deuterium Exchange (HDX)-MS (CAT#: STEM-B-0382-CJ)

At its core, HDX-MS measures the rate of solvent exchange—the phenomenon whereby atoms comprising the solvent are swapped for atoms of the protein (and vice versa). HDX-MS tracks solvent exchange by exposing the hydrogens within a protein molecule to a solvent containing a heavier hydrogen isotope—deuterium. The mass of a deuterium isotope is approximately 2.0141 Da, which is approximately double the mass of hydrogen (∼1.0080 Da), meaning that a solvent exchange event results in a small increase in the mass of a molecule. A 1 Da increase in mass is miniscule when compared with the mass of a protein; however, detecting and locating this increase in mass is eminently achievable with modern mass spectrometry equipment. By exposing proteins to a deuterated solvent for a defined period, quenching the solvent exchange reaction, and then measuring the increase in mass with a mass spectrometer, it is possible to determine solvent uptake progress over time. While solvent exchange is possible on all the labile hydrogens of a protein (i.e., any of the -NH, -OH, or -SH groups), only the peptide backbone amide hydrogens of the protein are tracked in a typical HDX-MS experiment. There are several benefits to this—peptide backbone hydrogens are uniformly distributed throughout the protein (save for proline-rich areas) and form secondary structure elements through hydrogen bonding.

Biopharmaceutica

1. Initial Sample Screening
2. Hydrogen Deuterium eXchange (HDX-MS): When diluted in heavy water (D2O), backbone hydrogens of amino acids exchange with deuterium at varying kinetics rates depending upon their hydrogen bonding and solvent accessibility. When the antigen is complexed with the antibody, this deuterium uptake rate is altered in the epitope regions. These differences can be measured accurately at various time points.
3. HDX-MS data analysis and interpretation.

• Sample: Protein monomers, Fragments and Aggregates
• Equipment: D2O, MS, RP-HPLC, Electron transfer dissociation (ETD)