PLIMSTEX (protein ligand interactions by mass spectrometry, tiration, and H/D exchange) is a new amide-exchange, mass spectrometric method for determining the affinity constants for protein/ligand interactions and for quantifying the conformational changes associated with ligand binding to proteins. It provides solution, not gas-phase, properties by taking advantage of ESI and MALDI mass spectrometry to measure accurately the mass of a protein as it undergoes exchange. The approach sidesteps the problem of relating gas-phase abundances to solution concentrations. Its validity has been tested with four equilibrium systems involving fatty-acid binding protein, calmodulin, and ras. Equilibrium constants determined with PLIMSTEX agree with literature values within a factor of six. Combined with kinetic measurements of H/D exchange, PLIMSTEX provides new insights on protein interactions with metal ions, peptides, and fatty acids. The kinetic approach is well suited to explore protein/protein interactions (e.g., self association of insulins) and peptide/peptide interactions (e.g., between membrane bound peptides such as gramicidin). Protein/metal ion interactions are evaluated in the presence of other metal ions and at various ionic strengths and show that affinities are stongly dependent on media. Those interactions can be probed rapidly and at concentrations in the nanomolar range. We expect the resolution of the method to be improved considerably when combined with protein digestion to peptides or by MS/MS fragmentation.
Back to Protein Characterization by Mass Spectrometry II
Back to The 56th Southeast Regional Meeting 2004 (November 10-13, 2004)