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 ␤-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. (J Am Soc Mass

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry

“…It is also worth mentioning here that HDX-MS can be applied to high-throughput screening of protein-ligand interactions and determination of binding affinity [48]. Methods, such as SUPREX (stability of unpurified proteins from rates of H/D exchange) [49 ,50-54] and PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange) [55][56][57][58], utilize the sensitivity of HDX rates to perturbations in protein structure by denaturing agents and/or ligand binding. In a typical SUPREX setting, a series of protein or proteinligand complex samples is incubated for a certain time in a deuterated buffer containing increasing amount of a chemical denaturant, for example, guanidinium hydrochloride.…”

“…Recently, a very similar method in which labile deuterium labeling is substituted with stable oxidative labeling of methionine residues has been proposed -SPROX (stability of proteins from rates of oxidation) [48,[60][61][62]. In PLIMSTEX, the protein is titrated with ligand under native conditions and subjected to HDX for a certain, optimized time [48,[55][56][57][58]. The resulting dependence of deuterium uptake on the added ligand concentration is used to determine the binding affinity.…”

Emerging mass spectrometry-based approaches to probe protein–receptor interactions: Focus on overcoming physiological barriers

“…In addition to structural information on protein drug/target binding, HDX MS methodology can also be used to determine binding affinity (57). Furthermore, HDX MS can also be used to identify protein segments involved in binding to other biopolymers, such as nucleic acids (58), indicating that this technique can become a truly invaluable tool in designing new and enhancing existing biopharmaceutical products including both protein- and oligonucleotide-based drugs and drug candidates.…”

Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

“…Furthermore, in addition to structural information on protein drug/target binding, HDX MS methodology can also be used to determine binding affinity (Tu et al 2010). …”