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

• DOI: 10.1007/s13361-010-0060-1
• Publication Date: 2011-01-31T14:41:36Z
• Authors: Patrick D. DeArmond; Graham M. West; Hai-Tsang Huang; Michael C. Fitzge...
• Keywords: 0301 basic medicine; Protein Folding; Calcineurin; Molecular Sequence Data; Proteins; Oxygen Isotopes; Mass Spectrometry; 03 medical and health sciences; Isotope Labeling; Cyclosporine; Thermodynamics; Amino Acid Sequence; Cyclophilin A; Oxidation-Reduction; Protein Binding

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)O(2) and H(2) (18)O(2) 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 (18)O/(16)O 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).