Protein-ligand binding studied by amide hydrogen exchange and mass spectrometry

The rates at which hydrogens located at peptide amide linkages in proteins undergo isotopic exchange when proteins are incubated in D2O may be sensitive to their access to the aqueous solvent. If ligands shield the binding sites from the solvent, isotopic exchange of hydrogens located in the vicinity of the binding sites might be the basis of a method for locating ligand binding sites on proteins. Although successful applications of this approach have been reported, results of more recent studies indicate that structural changes occurring distant from the binding epitope may also change upon ligand binding, thus confounding interpretation of results. The suggestion is made here that isotopic exchange rates of a subset of amide hydrogens whose exchange rates depend primarily on the access of the amide hydrogens to the aqueous solvent might be more diagnostic for ligand binding sites. Fundamental mechanisms for amide hydrogen exchange and the protein fragmentation/mass spectrometry method for determining hydrogen exchange rates are reviewed with emphasis on kinetic measurements for determining isotopic exchange rate constants for the most rapidly exchanging peptide amide hydrogens in large proteins.