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

Publication ,  Journal Article
West, GM; Thompson, JW; Soderblom, EJ; Dubois, LG; Dearmond, PD; Moseley, MA; Fitzgerald, MC
Published in: Anal Chem
July 1, 2010

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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Published In

Anal Chem

DOI

EISSN

1520-6882

Publication Date

July 1, 2010

Volume

82

Issue

13

Start / End Page

5573 / 5581

Location

United States

Related Subject Headings

  • Ubiquitin
  • Trypsin
  • Temperature
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Ribonuclease, Pancreatic
  • Proteins
  • Protein Binding
  • Oxidation-Reduction
  • Molecular Sequence Data
  • Methionine
 

Citation

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West, G. M., Thompson, J. W., Soderblom, E. J., Dubois, L. G., Dearmond, P. D., Moseley, M. A., & Fitzgerald, M. C. (2010). Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal Chem, 82(13), 5573–5581. https://doi.org/10.1021/ac100465a
West, Graham M., J Will Thompson, Erik J. Soderblom, Laura G. Dubois, Patrick D. Dearmond, M Arthur Moseley, and Michael C. Fitzgerald. “Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.Anal Chem 82, no. 13 (July 1, 2010): 5573–81. https://doi.org/10.1021/ac100465a.
West GM, Thompson JW, Soderblom EJ, Dubois LG, Dearmond PD, Moseley MA, et al. Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal Chem. 2010 Jul 1;82(13):5573–81.
West, Graham M., et al. “Mass spectrometry-based thermal shift assay for protein-ligand binding analysis.Anal Chem, vol. 82, no. 13, July 2010, pp. 5573–81. Pubmed, doi:10.1021/ac100465a.
West GM, Thompson JW, Soderblom EJ, Dubois LG, Dearmond PD, Moseley MA, Fitzgerald MC. Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal Chem. 2010 Jul 1;82(13):5573–5581.
Journal cover image

Published In

Anal Chem

DOI

EISSN

1520-6882

Publication Date

July 1, 2010

Volume

82

Issue

13

Start / End Page

5573 / 5581

Location

United States

Related Subject Headings

  • Ubiquitin
  • Trypsin
  • Temperature
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Ribonuclease, Pancreatic
  • Proteins
  • Protein Binding
  • Oxidation-Reduction
  • Molecular Sequence Data
  • Methionine