Skip to main content
SpringerLink
Log in

Detection of Protein Glutathionylation

  • Protocol
  • First Online:
Two-Dimensional Electrophoresis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 519))

Summary

Recent studies indicate that protein glutathionylation is an important regulatory mechanism. The develop-ment of redox proteomics techniques to identify proteins undergoing glutathionylation has a key role in defining the importance of this post-translational modification, although the available methods are not yet comparable to those for the study of other modifications like phosphorylation. We describe here methods that have been successfully employed to identify in vitro glutathionylated proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Thornton, J.M. (1981) Disulphide bridges in globular proteins. J Mol Biol 151, 261–287.

    Article  PubMed  CAS  Google Scholar 

  2. Rietsch, A., and Beckwith, J. (1998) The genetics of disulfide bond metabolism. Annu Rev Genet 32, 163–184.

    Article  PubMed  CAS  Google Scholar 

  3. Fahey, R.C., Hunt, J.S., and Windham, G.C. (1977) On the cysteine and cystine content of proteins: differences between intracellular and extracellular proteins. J Mol Evol 10, 155–160.

    Article  PubMed  CAS  Google Scholar 

  4. Saurin, A.T., Neubert, H., Brennan, J.P., and Eaton, P. (2004) Widespread sulfenic acid formation in tissues in response to hydrogen peroxide. Proc Natl Acad Sci U S A 101, 17982–17987.

    Article  PubMed  CAS  Google Scholar 

  5. Brennan, J.P., Wait, R., Begum, S., Bell, J.R., Dunn, M.J., and Eaton, P. (2004) Detection and mapping of widespread intermolecular protein disulfide formation during cardiac oxidative stress using proteomics with diagonal electrophoresis. J Biol Chem 279, 41352–41360.

    Article  PubMed  CAS  Google Scholar 

  6. Fratelli, M., Demol, H., Puype, M., Casagrande, S., Villa, P., Eberini, I., et al (2003) Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells. Proteomics 3, 1154–1161.

    Article  PubMed  CAS  Google Scholar 

  7. Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., et al (2002) Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci U. S. A. 99, 3505–3510.

    Article  PubMed  CAS  Google Scholar 

  8. Ghezzi, P., Bonetto, V., and Fratelli, M. (2005) Thiol-disulfide balance: From the concept of oxidative stress to that of redox regulation. Antioxid Redox Signal 7, 964–972.

    Article  PubMed  CAS  Google Scholar 

  9. Bursell, S.E., and King, G.L. (2000) The potential use of glutathionyl hemoglobin as a clinical marker of oxidative stress. Clin Chem 46, 145–146.

    PubMed  CAS  Google Scholar 

  10. Johnson, R.M., Buck, S., Schneider, H., Sampaio, I., Gage, D.A., Shen, T.L., et al (1997) Identification of pre-γ-globin. Hemoglobin 21, 143–153.

    Article  PubMed  CAS  Google Scholar 

  11. Mawatari, S., and Murakami, K. (2004) Different types of glutathionylation of hemoglobin can exist in intact erythrocytes. Arch Biochem Biophys 421, 108–114.

    Article  PubMed  CAS  Google Scholar 

  12. Murakami, K., and Mawatari, S. (2003) Oxidation of hemoglobin to methemoglobin in intact erythrocyte by a hydroperoxide induces formation of glutathionyl hemoglobin and binding of α-hemoglobin to membrane. Arch Biochem Biophys 417, 244–250.

    Article  PubMed  CAS  Google Scholar 

  13. Naito, C., Kajita, M., and Niwa, T. (1999) Determination of glutathionyl hemoglobin in hemodialysis patients using electrospray ionization liquid chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl 731, 121–124.

    Article  PubMed  CAS  Google Scholar 

  14. Niwa, T., Naito, C., Mawjood, A.H., and Imai, K. (2000) Increased glutathionyl hemoglobin indiabetes mellitus and hyperlipidemia demonstrated by liquid chromatography/electrospray ionization-mass spectrometry. Clin Chem 46, 82–88.

    PubMed  CAS  Google Scholar 

  15. Naito, C., and Niwa, T. (2000) Analysis of glutathionyl hemoglobin levels in diabetic patients by electrospray ionization liquid chromatography-mass spectrometry: Effect of vitamin E administration. J Chromatogr B Biomed Sci Appl 746, 91–94.

    Article  PubMed  CAS  Google Scholar 

  16. Takayama, F., Tsutsui, S., Horie, M., Shimokata, K., and Niwa, T. (2001) Glutathionyl hemoglobin in uremic patients undergoing hemodialysis and continuous ambulatory peritoneal dialysis. Kidney Int Suppl 78, S155–S158.

    Article  PubMed  CAS  Google Scholar 

  17. Piemonte, F., Pastore, A., Tozzi, G., Tagliacozzi, D., Santorelli, F.M., Carrozzo, R., et al (2001) Glutathione in blood of patients with Friedreich’s ataxia. Eur J Clin Invest 31, 1007–1011.

    Article  PubMed  CAS  Google Scholar 

  18. Sampathkumar, R., Balasubramanyam, M., Sudarslal, S., Rema, M., Mohan, V., and Balaram, P. (2005) Increased glutathionylated hemoglobin (HbSSG) in type 2 diabetes subjects with microangiopathy. Clin Biochem 38, 892–899.

    Article  PubMed  CAS  Google Scholar 

  19. Pastore, A., Mozzi, A.F., Tozzi, G., Gaeta, L.M., Federici, G., Bertini, E., et al (2003) Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography. Anal Biochem 312, 85–90.

    Article  PubMed  CAS  Google Scholar 

  20. Al-Abed, Y., VanPatten, S., Li, H., Lawson, J.A., FitzGerald, G.A., Manogue, K.R., and Bucala, R. (2001) Characterization of a novel hemoglobin-glutathione adduct that is elevated in diabetic patients. Mol Med 7, 619–623.

    PubMed  CAS  Google Scholar 

  21. Biroccio, A., Urbani, A., Massoud, R., di Ilio, C., Sacchetta, P., Bernardini, S., et al (2005) A quantitative method for the analysis of glycated and glutathionylated hemoglobin by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Anal Biochem 336, 279–288.

    Article  PubMed  CAS  Google Scholar 

  22. Caplan, J.F., Filipenko, N.R., Fitzpatrick, S.L., and Waisman, D.M. (2004) Regulation of annexin A2 by reversible glutathionylation. J Biol Chem 279, 7740–7750.

    Article  PubMed  CAS  Google Scholar 

  23. Sadidi, M., Geddes, T.J., and Kuhn, D.M. (2005) S-thiolation of tyrosine hydroxylase by reactive nitrogen species in the presence of cysteine or glutathione. Antioxid Redox Signal 7, 863–869.

    Article  PubMed  CAS  Google Scholar 

  24. Chen, F.C., and Ogut, O. (2006) Decline of contractility during ischemia-reperfusion injury: Actin glutathionylation and its effect on allosteric interaction with tropomyosin. Am J Physiol Cell Physiol 290, C719–C727.

    Article  PubMed  CAS  Google Scholar 

  25. Clavreul, N., Bachschmid, M.M., Hou, X., Shi, C., Idrizovic, A., Ido, Y., et al (2006) S-glutathiolation of p21ras by peroxynitrite mediates endothelial insulin resistance caused by oxidized low-density lipoprotein. Arterioscler Thromb Vasc Biol 26, 2454–2461.

    Article  PubMed  CAS  Google Scholar 

  26. Pastore, A., Tozzi, G., Gaeta, L.M., Bertini, E., Serafini, V., Di Cesare, S., et al (2003) Actin glutathionylation increases in fibroblasts of patients with Friedreich’s ataxia: a potential role in the pathogenesis of the disease. J Biol Chem 278, 42588–42595.

    Article  PubMed  CAS  Google Scholar 

  27. Townsend, D.M., Findlay, V.J., Fazilev, F., Ogle, M., Fraser, J., Saavedra, J.E., et al (2006) A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins. Mol Pharmacol 69, 501–508.

    Article  PubMed  CAS  Google Scholar 

  28. Cheng, G., Ikeda, Y., Iuchi, Y., and Fujii, J. (2005) Detection of S-glutathionylated proteins by glutathione S-transferase overlay. Arch Biochem Biophys 435, 42–49.

    Article  PubMed  CAS  Google Scholar 

  29. Eaton, P., Wright, N., Hearse, D.J., and Shattock, M.J. (2002) Glyceraldehyde phosphate dehydrogenase oxidation during cardiac ischemia and reperfusion. J Mol Cell Cardiol 34, 1549–1560.

    Article  PubMed  CAS  Google Scholar 

  30. Niture, S.K., Velu, C.S., Bailey, N.I., and Srivenugopal, K.S. (2005) S-thiolation mimicry: quantitative and kinetic analysis of redox status of protein cysteines by glutathione-affinity chromatography. Arch Biochem Biophys 444, 174–184.

    Article  PubMed  CAS  Google Scholar 

  31. Lind, C., Gerdes, R., Hamnell, Y., Schuppe-Koistinen, I., von Löwenhielm, H.B., Holmgren, A., and Cotgreave, I.A. (2002) Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch Biochem Biophys 406, 229–240.

    Article  PubMed  CAS  Google Scholar 

  32. Chai, Y.C., Hoppe, G., and Sears, J. (2003) Reversal of protein S-glutathiolation by glutaredoxin in the retinal pigment epithelium. Exp Eye Res 76, 155–159.

    Article  PubMed  CAS  Google Scholar 

  33. Eaton, P., Byers, H.L., Leeds, N., Ward, M.A., and Shattock, M.J. (2002) Detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. J Biol Chem 277, 9806–9811.

    Article  PubMed  CAS  Google Scholar 

  34. Sullivan, D.M., Wehr, N.B., Fergusson, M.M., Levine, R.L., and Finkel, T. (2000) Identification of oxidant-sensitive proteins: TNF-α induces protein glutathiolation. Biochemistry 39, 11121–11128.

    Article  PubMed  CAS  Google Scholar 

  35. Rokutan, K., Thomas, J.A., and Johnston, R.B. Jr. (1991) Phagocytosis and stimulation of the respiratory burst by phorbol diester initiate S-thiolation of specific proteins in macrophages. J Immunol 147, 260–264.

    PubMed  CAS  Google Scholar 

  36. Thomas, J.A., Park, E.M., Chai, Y.C., Brooks, R., Rokutan, K., and Johnston, R.B. Jr. (1991) S-thiolation of protein sulfhydryls. Adv Exp Med Biol 283, 95–103.

    PubMed  CAS  Google Scholar 

  37. Sanchez, J.-C., and Hochstrasser, D.F. 1998. High-resolution, IPG based, mini two-dimensional gel electrophoresis. In 2-D Proteome Analysis Protocols. (ed. A.J. Link). Humana Press, Totowa, NJ, pp. 227–233.

    Chapter  Google Scholar 

  38. Ghezzi, P., Romines, B., Fratelli, M., Eberini, I., Gianazza, E., Casagrande, S., et al (2002) Protein glutathionylation: coupling and uncoupling of glutathione to protein thiol groups in lymphocytes under oxidative stress and HIV infection. Mol Immunol 38, 773–780.

    Article  PubMed  CAS  Google Scholar 

  39. Kosower, N.S., and Kosower, E.M. (1987) Formation of disulfides with diamide. Methods Enzymol 143, 264–270.

    Article  PubMed  CAS  Google Scholar 

  40. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  41. Russell, W.K., Park, Z.Y., and Russell, D.H. (2001) Proteolysis in mixed organic-aqueous solvent systems: applications for peptide mass mapping using mass spectrometry. Anal Chem 73, 2682–2685.

    Article  PubMed  CAS  Google Scholar 

  42. Casagrande, S., Bonetto, V., Fratelli, M., Gianazza, E., Eberini, I., Massignan, T., et al (2002) Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thioredoxin systems. Proc Natl Acad Sci U. S. A. 99, 9745–9749.

    Article  PubMed  CAS  Google Scholar 

  43. Rossi, R., Cardaioli, E., Scaloni, A., Amiconi, G., and Di Simplicio, P. (1995) Thiol groups in proteins as endogenous reductants to determine glutathione-protein mixed disulphides in biological systems. Biochim Biophys Acta 1243, 230–238.

    PubMed  Google Scholar 

  44. Gmunder, H., Eck, H.P., Benninghoff, B., Roth, S., and Droge, W. (1990) Macrophages regulate intracellular glutathione levels of lymphocytes: evidence for an immunoregulatory role of cysteine. Cell Immunol 129, 32–46.

    Article  PubMed  CAS  Google Scholar 

  45. Gianazza, E., Giacon, P., Sahlin, B., and Righetti, P.G. (1985) Non-linear pH courses with immobilized pH gradients. Electrophoresis 6, 53–56.

    Article  CAS  Google Scholar 

  46. Gianazza, E. 2002. Casting immobilized pH gradients. In The Protein Protocol Handbook, 2nd ed. (ed. J.M. Walker). Humana Press, Totowa, NJ, pp. 169–180.

    Chapter  Google Scholar 

  47. Gianazza, E., Rocco, A.G., Marchetto, A., and Vergani, L. (2007) Immobilized pH gradients with electrodic plateaus (and other unusual procedures for 2DE). Electrophoresis 28, 2953–2956.

    Article  PubMed  CAS  Google Scholar 

  48. Hames, B.D. (1990) One-dimensional polyacrylamide gel electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach. (eds. B.D. Hames, and D. Rickwood). IRL, Oxford, pp. 1–147.

    Google Scholar 

  49. Gianazza, E., Astrua-Testori, S., Giacon, P., and Righetti, P.G. (1985) An improved protocol for 2D maps of serum proteins with immobilized pH gradients in the first dimension. Electrophoresis 6, 332–339.

    Article  CAS  Google Scholar 

  50. Skinner, M.K., and Griswold, M.D. (1983) Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures. Biochem J 209, 281–284.

    PubMed  CAS  Google Scholar 

  51. Laskey, R.A. (1980) The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol 65, 363–371.

    Article  PubMed  CAS  Google Scholar 

  52. de Souza, G.A., Godoy, L.M., Teixeira, V.R., Otake, A.H., Sabino, A., Rosa, J.C., Dinarte, A.R., Pinheiro, D.G., Silva, W.A., Jr., Eberlin, M.N., et al (2006) Proteomic and SAGE profiling of murine melanoma progression indicates the reduction of proteins responsible for ROS degradation. Proteomics 6, 1460–1470.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Work supported in part by AFM and by MIUR (FIRB 2003), to EG, and Fondazione CARIPLO, Milano (to PG).

Author information

Authors and Affiliations

  1. “Mario Negri” Institute for Pharmaceutical Research, via Eritrea 62, 20157, Milan, Italy

    Elisabetta Gianazza, Ivano Eberini & Pietro Ghezzi

Authors
  1. Elisabetta Gianazza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivano Eberini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pietro Ghezzi
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

    Rights and permissions

    Reprints and permissions

    Copyright information

    © 2009 Humana Press, a part of Springer Science+Business Media, LLC

    About this protocol

    Cite this protocol

    Gianazza, E., Eberini, I., Ghezzi, P. (2009). Detection of Protein Glutathionylation. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_26

    Download citation

    • DOI: https://doi.org/10.1007/978-1-59745-281-6_26

    • Published:

    • Publisher Name: Humana Press

    • Print ISBN: 978-1-58829-937-6

    • Online ISBN: 978-1-59745-281-6

    • eBook Packages: Springer Protocols

    Publish with us

    Policies and ethics

    Search

    Navigation