Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.
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.
Type
Journal articleSubject
Amino Acid SequenceAnimals
Carbonic Anhydrase II
Cattle
Chromatography, Liquid
Cyclophilin A
Hydrogen Peroxide
Ligands
Methionine
Molecular Sequence Data
Oxidation-Reduction
Protein Binding
Proteins
Ribonuclease, Pancreatic
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Temperature
Trypsin
Ubiquitin
Permalink
https://hdl.handle.net/10161/3996Published Version (Please cite this version)
10.1021/ac100465aPublication Info
West, GM; Thompson, JW; Soderblom, EJ; Dubois, LG; Moseley, MA; & Fitzgerald, MC (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.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
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 prote
Martin Arthur Moseley III
Adjunct Professor in the Department of Cell Biology
Erik James Soderblom
Associate Research Professor of Cell Biology
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 Princi
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