This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein-ligand interactions by mass spectrometry, titration, and H/D exchange (PLIMSTEX) is a mass spectrometric method for detg. assocn. consts. and binding stoichiometry for interactions of proteins with various ligands, as well as for quantifying the conformational changes assocd. with ligand binding to proteins. The assocn. consts. detd. with PLIMSTEX agree with literature values within a factor of six, establishing its validity for protein interactions involving metal ions, small org. mols., peptides, and proteins. PLIMSTEX provides soln., not gas-phase, properties by taking advantage of ESI and MALDI mass spectrometry to measure accurately the mass of a protein as it undergoes amide H/D exchange. The approach sidesteps the problem of relating gas-phase abundances of the protein or protein-ligand complex ions to their soln. concns. With on-column concn. and desalting, high picomole quantities of proteins are sufficient for reproducible mass detection, and the concn. of the protein can be as low as 10-8 M. It is amenable to different protein/ligand systems in physiol. relevant media. No specially labeled protein or ligand is needed. PLIMSTEX offers minimal perturbation of the binding equil. because it uses no denaturants, no addnl. spectroscopy or reaction probes, and no phys. sepn. of ligand and protein during binding. In this research, we are developing, testing, and extending further the method.
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