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dc.contributor.author West, GM
dc.contributor.author Thompson, JW
dc.contributor.author Soderblom, EJ
dc.contributor.author Dubois, LG
dc.contributor.author Dearmond, PD
dc.contributor.author Moseley, MA
dc.contributor.author Fitzgerald, MC
dc.coverage.spatial United States
dc.date.accessioned 2011-06-21T17:22:07Z
dc.date.issued 2010-07-01
dc.identifier http://www.ncbi.nlm.nih.gov/pubmed/20527820
dc.identifier.citation Anal Chem, 2010, 82 (13), pp. 5573 - 5581
dc.identifier.uri http://hdl.handle.net/10161/3996
dc.description.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.
dc.format.extent 5573 - 5581
dc.language eng
dc.language.iso en_US en_US
dc.relation.ispartof Anal Chem
dc.relation.isversionof 10.1021/ac100465a
dc.subject Amino Acid Sequence
dc.subject Animals
dc.subject Carbonic Anhydrase II
dc.subject Cattle
dc.subject Chromatography, Liquid
dc.subject Cyclophilin A
dc.subject Hydrogen Peroxide
dc.subject Ligands
dc.subject Methionine
dc.subject Molecular Sequence Data
dc.subject Oxidation-Reduction
dc.subject Protein Binding
dc.subject Proteins
dc.subject Ribonuclease, Pancreatic
dc.subject Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
dc.subject Temperature
dc.subject Trypsin
dc.subject Ubiquitin
dc.title Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.
dc.title.alternative en_US
dc.type Journal Article
dc.description.version Version of Record en_US
duke.date.pubdate 2010-7-1 en_US
duke.description.endpage 5581 en_US
duke.description.issue 13 en_US
duke.description.startpage 5573 en_US
duke.description.volume 82 en_US
dc.relation.journal Analytical Chemistry en_US
pubs.author-url http://www.ncbi.nlm.nih.gov/pubmed/20527820
pubs.issue 13
pubs.organisational-group /Duke
pubs.organisational-group /Duke/School of Medicine
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments/Biochemistry
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments/Cell Biology
pubs.organisational-group /Duke/School of Medicine/Basic Science Departments/Pharmacology & Cancer Biology
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments/Medicine
pubs.organisational-group /Duke/School of Medicine/Clinical Science Departments/Medicine/Medicine, Cardiology
pubs.organisational-group /Duke/Trinity College of Arts & Sciences
pubs.organisational-group /Duke/Trinity College of Arts & Sciences/Chemistry
pubs.publication-status Published
pubs.volume 82
dc.identifier.eissn 1520-6882

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