Skip to main content
Duke University Libraries
DukeSpace Scholarship by Duke Authors
  • Login
  • Ask
  • Menu
  • Login
  • Ask a Librarian
  • Search & Find
  • Using the Library
  • Research Support
  • Course Support
  • Libraries
  • About
View Item 
  •   DukeSpace
  • Duke Scholarly Works
  • Scholarly Articles
  • View Item
  •   DukeSpace
  • Duke Scholarly Works
  • Scholarly Articles
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

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

Thumbnail
View / Download
611.1 Kb
Date
2010-07-01
Authors
West, GM
Thompson, JW
Soderblom, EJ
Dubois, LG
Moseley, MA
Fitzgerald, MC
Repository Usage Stats
340
views
652
downloads
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 article
Subject
Amino Acid Sequence
Animals
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/10161/3996
Published Version (Please cite this version)
10.1021/ac100465a
Publication 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.
Collections
  • Scholarly Articles
More Info
Show full item record

Scholars@Duke

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 prote
Moseley

Martin Arthur Moseley III

Adjunct Professor in the Department of Cell Biology

Erik James Soderblom

Associate Research Professor of Cell Biology
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 Princi
Alphabetical list of authors with Scholars@Duke profiles.
Open Access

Articles written by Duke faculty are made available through the campus open access policy. For more information see: Duke Open Access Policy

Rights for Collection: Scholarly Articles


Works are deposited here by their authors, and represent their research and opinions, not that of Duke University. Some materials and descriptions may include offensive content. More info

Make Your Work Available Here

How to Deposit

Browse

All of DukeSpaceCommunities & CollectionsAuthorsTitlesTypesBy Issue DateDepartmentsAffiliations of Duke Author(s)SubjectsBy Submit DateThis CollectionAuthorsTitlesTypesBy Issue DateDepartmentsAffiliations of Duke Author(s)SubjectsBy Submit Date

My Account

LoginRegister

Statistics

View Usage Statistics
Duke University Libraries

Contact Us

411 Chapel Drive
Durham, NC 27708
(919) 660-5870
Perkins Library Service Desk

Digital Repositories at Duke

  • Report a problem with the repositories
  • About digital repositories at Duke
  • Accessibility Policy
  • Deaccession and DMCA Takedown Policy

TwitterFacebookYouTubeFlickrInstagramBlogs

Sign Up for Our Newsletter
  • Re-use & Attribution / Privacy
  • Harmful Language Statement
  • Support the Libraries
Duke University