Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

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 148–161 (2013)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Methionine oxidation in the SPROX protocol.
Figure 2: Schematic representation of the experimental workflow.
Figure 3: Distributions of the N1-normalized reporter ion values for two different iTRAQ tags obtained using the non-methionine-containing peptides from a (+) ligand SPROX sample.
Figure 4: Expected SPROX results from iTRAQ data analysis.
Figure 5: Distribution of the N2-normalized iTRAQ reporter ion value differences observed between the (+) and (−) ligand samples in a proteome-wide SPROX experiment in which the proteins in a yeast cell lysate were analyzed for binding to tamoxifen.
Figure 6: Chemical denaturation data sets from a proteome-wide SPROX experiment in which the proteins in a yeast cell lysate were analyzed for binding to tamoxifen.

Similar content being viewed by others

References

  1. West, G.M., Tang, L. & Fitzgerald, M.C. Thermodynamic analysis of protein stability and ligand binding using a chemical modification- and mass spectrometry-based strategy. Anal. Chem. 80, 4175–4185 (2008).

    Article  CAS  Google Scholar 

  2. Ghaemmaghami, S., Fitzgerald, M.C. & Oas, T.G. A quantitative, high-throughput screen for protein stability. Proc. Natl. Acad. Sci. USA 97, 8296–8301 (2000).

    Article  CAS  Google Scholar 

  3. Powell, K.D. & Fitzgerald, M.C. Accuracy and precision of a new H/D exchange- and mass spectrometry-based technique for measuring the thermodynamic properties of protein-peptide complexes. Biochemistry 42, 4962–4670 (2003).

    Article  CAS  Google Scholar 

  4. West, G.M. et al. Quantitative proteomics approach for identifying protein-drug interactions in complex mixtures using protein stability measurements. Proc. Natl. Acad. Sci. USA 107, 9078–9082 (2010).

    Article  CAS  Google Scholar 

  5. Dearmond, P.D., Xu, Y., Strickland, E.C., Daniels, K.G. & Fitzgerald, M.C. Thermodynamic analysis of protein-ligand interactions in complex biological mixtures using a shotgun proteomics approach. J. Proteome Res. 10, 4948–4958 (2011).

    Article  CAS  Google Scholar 

  6. Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245–246 (1989).

    Article  CAS  Google Scholar 

  7. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  Google Scholar 

  8. Lomenick, B. et al. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl. Acad. Sci. USA 106, 21984–21989 (2009).

    Article  CAS  Google Scholar 

  9. Liu, P.F., Kihara, D. & Park, C. Energetics-based discovery of protein-ligand interactions on a proteomic scale. J. Mol. Biol. 408, 147–162 (2011).

    Article  Google Scholar 

  10. DeArmond, P.D., West, G.M., Huang, H.T. & Fitzgerald, M.C. Stable isotope labeling strategy for protein-ligand binding analysis in multi-component protein mixtures. J. Am. Soc. Mass Spectrom. 22, 418–430 (2011).

    Article  CAS  Google Scholar 

  11. West, G.M. et al. Mass spectrometry-based thermal shift assay for protein-ligand binding analysis. Anal. Chem. 82, 5573–5581 (2010).

    Article  CAS  Google Scholar 

  12. Nozaki, Y. The preparation of guanidine hydrochloride. Methods Enzymol. 26, 43–50 (1972).

    Article  CAS  Google Scholar 

  13. Pace, C.N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131, 266–280 (1986).

    Article  CAS  Google Scholar 

  14. Noble, R.W. & Gibson, Q.H. The reaction of ferrous horseradish peroxidase with hydrogen peroxide. J. Biol. Chem. 245, 2409–2413 (1970).

    Article  CAS  Google Scholar 

  15. Shen, M. et al. Isolation and isotope labeling of cysteine- and methionine-containing tryptic peptides: application to the study of cell surface proteolysis. Mol. Cell Proteomics 2, 315–324 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

  1. Graham M West, Patrick D DeArmond & Ying Xu

    Present address: 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.).,

Authors and Affiliations

  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

Authors
  1. Erin C Strickland
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Ariel Geer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Duc T Tran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jagat Adhikari
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Graham M West
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrick D DeArmond
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ying Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michael C Fitzgerald
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Michael C Fitzgerald.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

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. (XLSX 6251 kb)

Supplementary Data 2

Runcompare input file used as a sample data set as described in Steps 44–46 (TXT 222 kb)

Supplementary Data 3

Runcompare input file used as a sample data set as described in Steps 44–46 (TXT 764 kb)

Supplementary Data 4

Output file generated from the sample data set as described in Steps 44–46 (TXT 303 kb)

Supplementary Data 5

The sample data set on which Steps 47–52 were performed ("Matched_NAD_Binding.xlsx") (XLSX 367 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Strickland, E., Geer, M., Tran, D. et al. Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation. Nat Protoc 8, 148–161 (2013). https://doi.org/10.1038/nprot.2012.146

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.146

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research