Protein-peptide affinity determination using an H/D exchange dilution strategy: Application to antigen-antibody interactions |
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Authors: | Tingting Tu Mihaela Drăgu§anu Brînduşa-Alina Petre Don L Rempel Michael Przybylski Michael L Gross |
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Institution: | (1) Department of Chemistry, Duke University, Durham, NC 27708-0346, USA;(2) Molecular Cardiology Research Institute, TUFTS-New England Medical Center, Boston, MA 02111, USA;(3) Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA |
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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. |
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