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Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions.

Publication ,  Journal Article
Meng, H; Ma, R; Fitzgerald, MC
Published in: Analytical chemistry
August 2018

Described here is a mass spectrometry-based proteomics approach for the large-scale analysis of protein-drug interactions. The approach involves the evaluation of ligand-induced protein folding free energy changes (ΔΔ Gf) using chemical denaturation and protein precipitation (CPP) to identify the protein targets of drugs and to quantify protein-drug binding affinities. This is accomplished in a chemical denaturant-induced unfolding experiment where the folded and unfolded protein fractions in each denaturant containing buffer are quantified by the amount of soluble or precipitated protein (respectively) that forms upon abrupt dilution of the chemical denaturant and subsequent centrifugation of the sample. In the proof-of-principle studies performed here, the CPP technique was able to identify the well-known protein targets of cyclosporin A and geldanamycin in a yeast. The technique was also used to identify protein targets of sinefungin, a broad-based methyltransferase inhibitor, in a human MCF-7 cell lysate. The CPP technique also yielded dissociation constant ( Kd) measurements for these well-studied drugs that were in general agreement with previously reported Kd or IC50 values. In comparison to a similar energetics-based technique, termed stability of proteins from rates of oxidation (SPROX), the CPP technique yielded significantly better (∼50% higher) proteomic coverage and a largely reduced false discovery rate.

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Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

August 2018

Volume

90

Issue

15

Start / End Page

9249 / 9255

Related Subject Headings

  • Thermodynamics
  • Proteomics
  • Proteins
  • Protein Denaturation
  • Protein Binding
  • Pharmaceutical Preparations
  • MCF-7 Cells
  • Lactams, Macrocyclic
  • Humans
  • Fungal Proteins
 

Citation

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Meng, H., Ma, R., & Fitzgerald, M. C. (2018). Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions. Analytical Chemistry, 90(15), 9249–9255. https://doi.org/10.1021/acs.analchem.8b01772
Meng, He, Renze Ma, and Michael C. Fitzgerald. “Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions.Analytical Chemistry 90, no. 15 (August 2018): 9249–55. https://doi.org/10.1021/acs.analchem.8b01772.
Meng H, Ma R, Fitzgerald MC. Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions. Analytical chemistry. 2018 Aug;90(15):9249–55.
Meng, He, et al. “Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions.Analytical Chemistry, vol. 90, no. 15, Aug. 2018, pp. 9249–55. Epmc, doi:10.1021/acs.analchem.8b01772.
Meng H, Ma R, Fitzgerald MC. Chemical Denaturation and Protein Precipitation Approach for Discovery and Quantitation of Protein-Drug Interactions. Analytical chemistry. 2018 Aug;90(15):9249–9255.
Journal cover image

Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

August 2018

Volume

90

Issue

15

Start / End Page

9249 / 9255

Related Subject Headings

  • Thermodynamics
  • Proteomics
  • Proteins
  • Protein Denaturation
  • Protein Binding
  • Pharmaceutical Preparations
  • MCF-7 Cells
  • Lactams, Macrocyclic
  • Humans
  • Fungal Proteins