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Protein-peptide affinity determination using an H/D exchange dilution strategy: Application to antigen-antibody interactions

  • Tingting Tu
  • Mihaela Drăgu§anu
  • Brînduşa-Alina Petre
  • Don L. Rempel
  • Michael Przybylski
  • Michael L. Gross
Focus: Affinity Mass Spectrometry
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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.

Keywords

Melittin Epitope Mapping Binding Stoichiometry Free Peptide Exchange Mass Spectrometry 
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Copyright information

© American Society for Mass Spectrometry 2010

Authors and Affiliations

  • Tingting Tu
    • 1
  • Mihaela Drăgu§anu
    • 2
  • Brînduşa-Alina Petre
    • 2
  • Don L. Rempel
    • 1
  • Michael Przybylski
    • 2
  • Michael L. Gross
    • 1
  1. 1.Center for Biomedical and Bioorganic Mass Spectrometry, Department of ChemistryWashington University in St. LouisSt. LouisUSA
  2. 2.Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of ChemistryUniversity of KonstanzKonstanzGermany

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