Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.

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2010-07-01

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Abstract

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.

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10.1021/ac100465a

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West, GM, JW Thompson, EJ Soderblom, LG Dubois, MA Moseley and MC Fitzgerald (2010). Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal Chem, 82(13). pp. 5573–5581. 10.1021/ac100465a Retrieved from https://hdl.handle.net/10161/3996.

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Scholars@Duke

Thompson

J. Will Thompson

Adjunct Assistant Professor in the Department of Pharmacology & Cancer Biology

Dr. Thompson's research focuses on the development and deployment of proteomics and metabolomics mass spectrometry techniques for the analysis of biological systems. He served as the Assistant Director of the Proteomics and Metabolomics Shared Resource in the Duke School of Medicine from 2007-2021. He currently maintains collaborations in metabolomics and proteomics research at Duke, and develops new tools for chemical analysis as a Principal Scientist at 908 Devices in Carrboro, NC.

Soderblom

Erik James Soderblom

Associate Research Professor of Cell Biology

Director, Proteomics and Metabolomics Core Facility

Moseley

Martin Arthur Moseley

Adjunct Professor in the Department of Cell Biology
Fitzgerald

Michael C. Fitzgerald

Professor of Chemistry

Dr. Fitzgerald’s research group is focused on studies of protein folding and function. The group utilizes a combination of covalent labeling strategies (e.g. protein amide H/D exchange and methionine oxidiation) and mass spectrometry techniques to investigate the thermodynamic properties of protein folding and ligand binding reactions. Current research efforts involve: (1) the development new biophysical methods that enable protein folding and stability measurements to be performed on the proteomic scale; and (2) the application of these new methods in the areas of disease detection, diagnosis, and therapy.


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