Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.
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Described here is a mass spectrometry-based screening assay for the detection of protein-ligand binding interactions in multicomponent protein mixtures. The assay utilizes an oxidation labeling protocol that involves using hydrogen peroxide to selectively oxidize methionine residues in proteins in order to probe the solvent accessibility of these residues as a function of temperature. The extent to which methionine residues in a protein are oxidized after specified reaction times at a range of temperatures is determined in a MALDI analysis of the intact proteins and/or an LC-MS analysis of tryptic peptide fragments generated after the oxidation reaction is quenched. Ultimately, the mass spectral data is used to construct thermal denaturation curves for the detected proteins. In this proof-of-principle work, the protocol is applied to a four-protein model mixture comprised of ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). The new protocol's ability to detect protein-ligand binding interactions by comparing thermal denaturation data obtained in the absence and in the presence of ligand is demonstrated using cyclosporin A (CsA) as a test ligand. The known binding interaction between CsA and CypA was detected using both the MALDI- and LC-MS-based readouts described here.
SubjectAmino Acid Sequence
Carbonic Anhydrase II
Molecular Sequence Data
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Published Version (Please cite this version)10.1021/ac100465a
Publication InfoDearmond, PD; Dubois, Laura; Fitzgerald, MC; Moseley, Martin Arthur III; Soderblom, Erik James; Thompson, JW; & West, GM (2010). Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal Chem, 82(13). pp. 5573-5581. 10.1021/ac100465a. Retrieved from http://hdl.handle.net/10161/3996.
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Associate Professor in Medicine
Assistant Research Professor of Cell Biology
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