Analytical Method for Protein Mapping using Hydrogen Deuterium Exchange

The present invention describes analytical methods for protein and peptide mapping based on hydrogen/deuterium exchange (HDX) with reduced or eliminated back-exchange of deuterium for hydrogen. The methods include the steps of (a) providing a peptide or protein comprising a solvent accessible hydrogen; (b) exchanging the solvent accessible hydrogen for a deuterium; (c) separating the peptide or protein with supercritical fluid chromatography; and (d) analyzing by mass spectrometry the mass of the separated peptide or protein. Supercritical fluid chromatography enables the observation of fast exchanging hydrogen atoms missed using conventional liquid chromatography methods. Using supercritical fluid chromatography instead of high performance liquid chromatography allows the faster exchanging hydrogen atoms previously missed in HDX experiments to now be observable.

The information obtained from analyzing the mass of the peptide or protein can be extremely useful in understanding protein confirmation, protein/protein interactions, and protein/ligand interactions as well as characterizing the folding pathway of proteins. This understanding can be used to build models on protein-ligand binding sites to be used in the pharmaceutical industry for intelligent drug design.