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

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2010
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Tu, Tingting
Drăguşanu, Mihaela
Rempel, Don L.
Gross, Michael L.
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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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ISO 690TU, Tingting, Mihaela DRĂGUŞANU, Brînduşa-Alina PETRE, Don L. REMPEL, Michael PRZYBYLSKI, Michael L. GROSS, 2010. Protein-peptide affinity determination using an H/D exchange dilution strategy : Application to antigen-antibody interactions. In: Journal of the American Society for Mass Spectrometry (JASMS). ACS Publications. 2010, 21(10), pp. 1660-1667. ISSN 1044-0305. eISSN 1879-1123. Available under: doi: 10.1021/jasms.8b03620
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@article{Tu2010Prote-1025.2,
  year={2010},
  doi={10.1021/jasms.8b03620},
  title={Protein-peptide affinity determination using an H/D exchange dilution strategy : Application to antigen-antibody interactions},
  number={10},
  volume={21},
  issn={1044-0305},
  journal={Journal of the American Society for Mass Spectrometry (JASMS)},
  pages={1660--1667},
  author={Tu, Tingting and Drăguşanu, Mihaela and Petre, Brînduşa-Alina and Rempel, Don L. and Przybylski, Michael and Gross, Michael L.}
}
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