title: Protein-peptide affinity determination using an h/d exchange dilution strategy: application to antigen-antibody interactions.
abstract: A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc.2003, 125, 5252-5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide β-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.
authors.affiliation: Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, USA.
authors.forename: Tingting
authors.initials: T
authors.lastname: Tu
authors.forename: Mihaela
authors.initials: M
authors.lastname: Drăguşanu
authors.forename: Brînduşa-Alina
authors.initials: BA
authors.lastname: Petre
authors.forename: Don L
authors.initials: DL
authors.lastname: Rempel
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods*; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D013552:Swine; D001615:beta-Endorphin / Q000737:chemistry / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002147:Calmodulin; D001615:beta-Endorphin
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