Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 22, Issue 3, Pages 418-430Publisher
SPRINGER
DOI: 10.1007/s13361-010-0060-1
Keywords
Protein folding; Ligand binding; Thermodynamics; Covalent labeling; Quantitative proteomics; Cyclophilin A; Cyclosporin A; Calcineurin
Funding
- National Institutes of Health [GM084174]
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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 O-18/O-16 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).
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