Research Article

Journal of The American Society for Mass Spectrometry

, Volume 22, Issue 3, pp 418-430

Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures

  • Patrick D. DeArmondAffiliated withDepartment of Chemistry, Duke University
  • , Graham M. WestAffiliated withDepartment of Chemistry, Duke University
  • , Hai-Tsang HuangAffiliated withDepartment of Chemistry, Duke University
  • , Michael C. FitzgeraldAffiliated withDepartment of Chemistry, Duke University Email author 

Abstract

Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H 2 16 O2 and H 2 18 O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

Key words

Protein folding Ligand binding Thermodynamics Covalent labeling Quantitative proteomics Cyclophilin A Cyclosporin A Calcineurin