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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Protein-peptide affinity determination using an h/d exchange dilution strategy: application to antigen-antibody
Tingting Tu1, Mihaela Drăguşanu, Brînduşa-Alina Petre
1Center for Biomedical and Bioorganic Mass Spectrometry, Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, USA.
Summary
A new dilution strategy for hydrogen/deuterium exchange mass spectrometry (HDX-MS) quantifies protein-peptide binding affinities. This dPLIMSTEX method accurately measures interactions, including antigen-antibody binding, offering a sensitive tool for affinity determination.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Mass Spectrometry
Background:
- Accurate quantification of protein-peptide interactions is crucial in biological research.
- Existing methods for determining binding affinity can be complex or limited in scope.
Purpose of the Study:
- To introduce and validate a novel dilution strategy for hydrogen/deuterium exchange mass spectrometry (HDX-MS) called dPLIMSTEX.
- To demonstrate the utility of dPLIMSTEX for quantifying protein-peptide and antigen-antibody binding affinities.
Main Methods:
- Utilized a dilution strategy (dPLIMSTEX) based on protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX).
- Employed mass spectrometry to measure the mass of peptide ligands as a readout for binding.
- Applied dPLIMSTEX to model systems: calmodulin-β-endorphin and a 3-nitrotyrosine peptide with a monoclonal antibody.
Main Results:
- dPLIMSTEX accurately determined binding affinity for the calmodulin-β-endorphin interaction, consistent with traditional PLIMSTEX and literature values.
- Quantified a low nanomolar dissociation constant for an antigen-antibody interaction.
- Confirmed a 1:2 antibody/peptide binding stoichiometry and identified a minimum five-amino acid epitope.
Conclusions:
- The dPLIMSTEX method provides a sensitive and powerful approach for quantitative determination of peptide affinities.
- This technique is a valuable complement to conventional immuno-analytical methods.
- dPLIMSTEX expands the capabilities of HDX-MS for studying molecular interactions.
