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.

  • Article
  • Published:

Quantitative reactivity profiling predicts functional cysteines in proteomes

Nature volume 468, pages 790–795 (2010)Cite this article

Subjects

Abstract

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

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: A quantitative approach to globally profile cysteine reactivity in proteomes.
Figure 2: Hyper-reactive cysteines are highly enriched in functional residues.
Figure 3: Functional characterization of the hyper-reactive cysteines in PRMT1.
Figure 4: Functional characterization of YHR122W/FAM96B.
Figure 5: Quantitative reactivity profiling predicts functional cysteines in designed proteins.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Eisenberg, D., Marcotte, E. M., Xenarios, I. & Yeates, T. O. Protein function in the post-genomic era. Nature 405, 823–826 (2000)

    Article  CAS  Google Scholar 

  2. Zhao, Y. & Jensen, O. N. Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques. Proteomics 9, 4632–4641 (2009)

    Article  CAS  Google Scholar 

  3. Bulaj, G., Kortemme, T. & Goldenberg, D. P. Ionization reactivity relationships for cysteine thiols in polypeptides. Biochemistry 37, 8965–8972 (1998)

    Article  CAS  Google Scholar 

  4. Giles, N. M., Giles, G. I. & Jacob, C. Multiple roles of cysteine in biocatalysis. Biochem. Biophys. Res. Commun. 300, 1–4 (2003)

    Article  CAS  Google Scholar 

  5. Reddie, K. G. & Carroll, K. S. Expanding the functional diversity of proteins through cysteine oxidation. Curr. Opin. Chem. Biol. 12, 746–754 (2008)

    Article  CAS  Google Scholar 

  6. Voss, A. A., Lango, J., Ernst-Russell, M., Morin, D. & Pessah, I. N. Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry. J. Biol. Chem. 279, 34514–34520 (2004)

    Article  CAS  Google Scholar 

  7. Lewis, C. T., Seyer, J. M. & Carlson, G. M. Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP). J. Biol. Chem. 264, 27–33 (1989)

    CAS  PubMed  Google Scholar 

  8. Knowles, J. R. Intrinsic pK a-values of functional-groups in enzymes: improper deductions from pH-dependence of steady-state parameters. CRC Crit. Rev. Biochem. 4, 165–173 (1976)

    Article  CAS  Google Scholar 

  9. Fomenko, D. E., Xing, W., Adair, B. M., Thomas, D. J. & Gladyshev, V. N. High-throughput identification of catalytic redox-active cysteine residues. Science 315, 387–389 (2007)

    Article  ADS  CAS  Google Scholar 

  10. Sethuraman, M. et al. Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures. J. Proteome Res. 3, 1228–1233 (2004)

    Article  CAS  Google Scholar 

  11. Baty, J. W., Hampton, M. B. & Winterbourn, C. C. Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells. Biochem. J. 389, 785–795 (2005)

    Article  CAS  Google Scholar 

  12. Salsbury, F. R., Jr, Knutson, S. T., Poole, L. B. & Fetrow, J. S. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid. Protein Sci. 17, 299–312 (2008)

    Article  CAS  Google Scholar 

  13. Leonard, S. E., Reddie, K. G. & Carroll, K. S. Mining the thiol proteome for sulfenic acid modifications reveals new targets for oxidation in cells. ACS Chem. Biol. 4, 783–799 (2009)

    Article  CAS  Google Scholar 

  14. Kim, J.-R., Yoon, H. W., Kwon, K.-S., Lee, S.-R. & Rhee, S. G. Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH. Anal. Biochem. 283, 214–221 (2000)

    Article  CAS  Google Scholar 

  15. Speers, A. E. & Cravatt, B. F. A tandem orthogonal proteolysis strategy for high-content chemical proteomics. J. Am. Chem. Soc. 127, 10018–10019 (2005)

    Article  CAS  Google Scholar 

  16. Weerapana, E., Simon, G. M. & Cravatt, B. F. Disparate proteome reactivity profiles of carbon electrophiles. Nature Chem. Biol. 4, 405–407 (2008)

    Article  CAS  Google Scholar 

  17. Dennehy, M. K., Richards, K. A., Wernke, G. R., Shyr, Y. & Liebler, D. C. Cytosolic and nuclear protein targets of thiol-reactive electrophiles. Chem. Res. Toxicol. 19, 20–29 (2006)

    Article  CAS  Google Scholar 

  18. Shin, N.-Y., Liu, Q., Stamer, S. L. & Liebler, D. C. Protein targets of reactive electrophiles in human liver microsomes. Chem. Res. Toxicol. 20, 859–867 (2007)

    Article  CAS  Google Scholar 

  19. Speers, A. E., Adam, G. C. & Cravatt, B. F. Activity-based protein profiling in vivo using a copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 4686–4687 (2003)

    Article  CAS  Google Scholar 

  20. Weerapana, E., Speers, A. E. & Cravatt, B. F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes. Nature Protocols 2, 1414–1425 (2007)

    Article  CAS  Google Scholar 

  21. Shiio, Y. & Aebersold, R. Quantitative proteome analysis using isotope-coded affinity tags and mass spectrometry. Nature Protocols 1, 139–145 (2006)

    Article  CAS  Google Scholar 

  22. Board, P. G. et al. Identification, characterization, and crystal structure of the omega class glutathione transferases. J. Biol. Chem. 275, 24798–24806 (2000)

    Article  CAS  Google Scholar 

  23. Thompson, S. et al. Mechanistic studies on β-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes. Biochemistry 28, 5735–5742 (1989)

    Article  CAS  Google Scholar 

  24. Iyer, L. M., Koonin, E. V. & Aravind, L. Novel predicted peptidases with a potential role in the ubiquitin signaling pathway. Cell Cycle 3, 1440–1450 (2004)

    Article  CAS  Google Scholar 

  25. Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D. & Liebler, D. C. Global analysis of protein damage by the lipid electrophile 4-hydroxy-2-nonenal. Mol. Cell. Proteomics 8, 670–680 (2009)

    Article  CAS  Google Scholar 

  26. Zhang, X. & Cheng, X. Structure of the predominant protein arginine methyltransferase PRMT1 and analysis of Its binding to substrate peptides. Structure 11, 509–520 (2003)

    Article  CAS  Google Scholar 

  27. Harrop, S. J. et al. Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-Å resolution. J. Biol. Chem. 276, 44993–45000 (2001)

    Article  CAS  Google Scholar 

  28. Hao, G., Derakhshan, B., Shi, L., Campagne, F. & Gross, S. S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures. Proc. Natl Acad. Sci. USA 103, 1012–1017 (2006)

    Article  ADS  CAS  Google Scholar 

  29. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002)

    Article  ADS  CAS  Google Scholar 

  30. Lill, R. Function and biogenesis of iron sulphur proteins. Nature 460, 831–838 (2009)

    Article  ADS  CAS  Google Scholar 

  31. Pierik, A. J., Netz, D. J. & Lill, R. Analysis of iron–sulfur protein maturation in eukaryotes. Nature Protocols 4, 753–766 (2009)

    Article  CAS  Google Scholar 

  32. Netz, D. J. A., Pierik, A. J., Stumpfig, M., Muhlenhoff, U. & Lill, R. The Cfd1–Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol. Nature Chem. Biol. 3, 278–286 (2007)

    Article  CAS  Google Scholar 

  33. Okerberg, E. S. et al. High-resolution functional proteomics by active-site peptide profiling. Proc. Natl Acad. Sci. USA 102, 4996–5001 (2005)

    Article  ADS  CAS  Google Scholar 

  34. Nazif, T. & Bogyo, M. Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors. Proc. Natl Acad. Sci. USA 98, 2967–2972 (2001)

    Article  ADS  CAS  Google Scholar 

  35. Chen, G. et al. Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling. J. Am. Chem. Soc. 125, 8130–8133 (2003)

    Article  CAS  Google Scholar 

  36. Silverman, J. A. & Harbury, P. B. Rapid mapping of protein structure, interactions, and ligand binding by misincorporation proton-alkyl exchange. J. Biol. Chem. 277, 30968–30975 (2002)

    Article  CAS  Google Scholar 

  37. Isom, D. G., Vardy, E., Oas, T. G. & Hellinga, H. W. Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity. Proc. Natl Acad. Sci. USA 107, 4908–4913 (2010)

    Article  ADS  CAS  Google Scholar 

  38. Kortemme, T. & Creighton, T. E. Ionisation of cysteine residues at the termini of model α-helical peptides. Relevance to unusual thiol pK a values in proteins of the thioredoxin family. J. Mol. Biol. 253, 799–812 (1995)

    Article  CAS  Google Scholar 

  39. Macpherson, L. J. et al. Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. Nature 445, 541–545 (2007)

    Article  ADS  CAS  Google Scholar 

  40. Eng, J. K., Mccormack, A. L. & Yates, J. R. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994)

    Article  CAS  Google Scholar 

  41. Kispal, G., Csere, P., Prohl, C. & Lill, R. The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. EMBO J. 18, 3981–3989 (1999)

    Article  CAS  Google Scholar 

  42. Tabb, D. L., McDonald, W. H. & Yates, J. R., III DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1, 21–26 (2002)

    Article  CAS  Google Scholar 

  43. Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nature Methods 4, 207–214 (2007)

    Article  CAS  Google Scholar 

  44. Collins, S. R. et al. Toward a comprehensive atlas of the physical interactome of Saccharomyces cerevisiae . Mol. Cell. Proteomics 6, 439–450 (2007)

    Article  CAS  Google Scholar 

  45. Pedrioli, P. G. A. et al. A common open representation of mass spectrometry data and its application to proteomics research. Nature Biotechnol. 22, 1459–1466 (2004)

    Article  CAS  Google Scholar 

  46. Park, S. K., Venable, J. D., Xu, T. & Yates, J. R. A quantitative analysis software tool for mass spectrometry-based proteomics. Nature Methods 5, 319–322 (2008)

    Article  CAS  Google Scholar 

  47. Vallee, B. L. & Hoch, F. L. Zinc, a component of yeast alcohol dehydrogenase. Proc. Natl Acad. Sci. USA 41, 327–338 (1955)

    Article  ADS  CAS  Google Scholar 

  48. Zanghellini, A. et al. New algorithms and an in silico benchmark for computational enzyme design. Protein Sci. 15, 2785–2794 (2006)

    Article  CAS  Google Scholar 

  49. Ma, S., Devi-Kesavan, L. S. & Gao, J. Molecular dynamics simulations of the catalytic pathway of a cysteine protease: a combined QM/MM study of human cathepsin K. J. Am. Chem. Soc. 129, 13633–13645 (2007)

    Article  CAS  Google Scholar 

  50. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science 319, 1387–1391 (2008)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank T. Bartfai, I. Wilson and members of the B.F.C. laboratory for comments and critical reading of the manuscript, T. Ji for experimental assistance and J. Gallaher for expression of designed proteins. This work was supported by the National Institutes of Health (CA087660, MH084512), a Pfizer Postdoctoral Fellowship (E.W.), a Koshland Graduate Fellowship in Enzyme Biochemistry (G.M.S.), a National Science Foundation predoctoral fellowship (D.A.B.) and the Skaggs Institute for Chemical Biology.

Author information

Author notes

  1. Eranthie Weerapana and Chu Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Eranthie Weerapana, Chu Wang, Gabriel M. Simon, Daniel A. Bachovchin & Benjamin F. Cravatt

  2. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Eranthie Weerapana, Chu Wang, Gabriel M. Simon, Myles B. D. Dillon, Daniel A. Bachovchin, Kerri Mowen & Benjamin F. Cravatt

  3. Department of Biochemistry, University of Washington, Seattle, 98195, Washington, USA

    Florian Richter, Sagar Khare & David Baker

  4. Interdisciplinary Program in Biomolecular Structure and Design, University of Washington, Seattle, 98195, Washington, USA

    Florian Richter & David Baker

  5. Howard Hughes Medical Institute, University of Washington, Seattle, 98195, Washington, USA

    Sagar Khare & David Baker

Authors
  1. Eranthie Weerapana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gabriel M. Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Florian Richter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sagar Khare
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Myles B. D. Dillon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Daniel A. Bachovchin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kerri Mowen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Benjamin F. Cravatt
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.F.C., E.W. and C.W. conceived the project and E.W. and C.W. performed MS experiments and yeast growth/Leu1 assays. C.W. and G.M.S. performed computational data analyses. S.K., F.R. and D.B. performed computational design of cysteine hydrolases and measured activity using a fluorogenic assay. D.A.B. purified PRMT1 and M.B.D.D. and K.M. performed PRMT1 activity assays. B.F.C., E.W., C.W. and G.M.S. analysed data and wrote the manuscript.

Corresponding author

Correspondence to Benjamin F. Cravatt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains a Supplementary Discussion, Supplementary Methods, Supplementary Figures 1-18 with legends, Supplementary Tables 1-8 and additional references. (PDF 6480 kb)

Supplementary Table

The file contains Supplementary Table 9. (XLS 7446 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Weerapana, E., Wang, C., Simon, G. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010). https://doi.org/10.1038/nature09472

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09472

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