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Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation

Nature Protocols volume 8, pages 148161 (2013) | Download Citation


The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (isobaric mass tags for relative and absolute quantification, or iTRAQ) with a covalent labeling technique, termed stability of proteins from rates of oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide–mediated oxidation of methionine residues as a function of the chemical denaturant (e.g., guanidine hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand-binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to the detection of both the on- and off-target effects of ligand binding. The protocol can be completed in 5 d.

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This work was supported in part by US National Institutes of Health grant no. GM084174 (to M.C.F.) and in part by National Science Foundation (NSF) grant no. CHE-0848462 (to M.C.F.). The NSF grant, which was made possible with funds from the American Recovery and Reinvestment Act (ARRA), is jointly funded by the Analytical and Surface Chemistry Program in the Chemistry Division at NSF and by the Biomolecular Systems Cluster in the Division of Molecular and Cellular Biosciences at NSF. The mass spectrometer system used in this work was purchased with funds from National Institutes of Health grant no. S10RR027746 (to M.C.F.).

Author information

Author notes

    • Graham M West
    • , Patrick D DeArmond
    •  & Ying Xu

    Present addresses: The Scripps Research Institute, Jupiter, Florida, USA (G.M.W.), US Environmental Protection Agency, Las Vegas, Nevada (P.D.D.), and Clark University, Worcester, Massachusetts, USA (Y.X.).


  1. Department of Chemistry, Duke University, Durham, North Carolina, USA.

    • Erin C Strickland
    • , M Ariel Geer
    • , Graham M West
    • , Patrick D DeArmond
    • , Ying Xu
    •  & Michael C Fitzgerald
  2. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA.

    • Duc T Tran
    • , Jagat Adhikari
    •  & Michael C Fitzgerald


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G.M.W., P.D.D., Y.X., E.C.S., M.A.G., D.T.T. and J.A. contributed to the development and optimization of the described SPROX protocol. E.C.S. collected and analyzed the SPROX data highlighted in this manuscript. E.C.S., M.A.G., D.T.T., J.A. and M.C.F. drafted the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael C Fitzgerald.

Supplementary information

Excel files

  1. 1.

    Supplementary Data 1

    A sample data set on which Steps 36–43 were performed ("Sample_Data_Set.xlsx"). The data set is from the NAD binding study described in reference 5.

  2. 2.

    Supplementary Data 5

    The sample data set on which Steps 47–52 were performed ("Matched_NAD_Binding.xlsx")

Text files

  1. 1.

    Supplementary Data 2

    Runcompare input file used as a sample data set as described in Steps 44–46

  2. 2.

    Supplementary Data 3

    Runcompare input file used as a sample data set as described in Steps 44–46

  3. 3.

    Supplementary Data 4

    Output file generated from the sample data set as described in Steps 44–46

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