Abstract
Protein cysteine sulfhydryl groups are susceptible to a number of redox-dependent modifications, including an interchange between the reduced sulfhydryl and an oxidized disulfide state. A growing body of evidence suggests that reversible disulfide bond formation alters the structure and function of proteins. In this chapter, a method is described for isolating disulfide bonded proteins from different subcellular compartments by using a differential detergent fractionation technique followed by sequential nonreducing/reducing two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (i.e., Redox 2D-PAGE). This method can be adapted to examine individual redox-active proteins by immunoprecipitating an epitope-tagged redox protein expressed in cultured cells and using Redox 2D-PAGE and mass spectrometry to identify proteins that form mixed disulfides with the tagged protein. With the use of these techniques, it is shown that disulfide bond formation occurs within families of cytoplasmic proteins and may provide a common mechanism used to control multiple physiological processes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sitia, R. and Molteni, S. N. (2004) Stress, protein (mis)folding, and signaling: the redox connection. Sci STKE 2004, pe27.
Rhee, S. G., Kang, S. W., Jeong, W., Chang, T. S., Yang, K. S., and Woo, H. A. (2005) Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr. Opin. Cell Biol. 17, 183–189.
Linke, K. and Jakob, U. (2003) Not every disulfide lasts forever: disulfide bond formation as a redox switch. Antioxid. Redox Signal 5, 425–434.
Sommer, A. and Traut, R. R. (1974) Diagonal polyacrylamide-dodecyl sulfate gel electrophoresis for the identification of ribosomal proteins crosslinked with methyl-4-mercaptobutyrimidate. Proc. Natl. Acad. Sci. USA 71, 3946–3950.
Molinari, M. and Helenius, A. (1999) Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells. Nature 402, 90–93.
Molinari, M. and Helenius, A. (2000) Chaperone selection during glycoprotein translocation into the endoplasmic reticulum. Science 288, 331–333.
Yano, H., Wong, J. H., Lee, Y. M., Cho, M. J., and Buchanan, B. B. (2001) A strategy for the identification of proteins targeted by thioredoxin. Proc. Natl. Acad. Sci. USA 98, 4794–4799.
Lindahl, M. and Florencio, F. J. (2003) Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different. Proc. Natl. Acad. Sci. USA 100, 16107–16112.
Cumming, R. C., Andon, N. L., Haynes, P. A., Park, M., Fischer, W. H., and Schubert, D. (2004) Protein disulfide bond formation in the cytoplasm during oxidative stress. J. Biol. Chem. 279, 21749–21758.
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.
Ramsby, M. L. and Makowski, G. S. (1999) Differential detergent fractionation of eukaryotic cells. Analysis by two-dimensional gel electrophoresis. Methods Mol. Biol. 112, 53–66.
Tan, S., Sagara, Y., Liu, Y., Maher, P., and Schubert, D. (1998) The regulation of reactive oxygen species production during programmed cell death. J. Cell Biol. 141, 1423–1432.
Tan, S., Somia, N., Maher, P., and Schubert, D. (2001) Regulation of antioxidant metabolism by translation initiation factor 2alpha. J. Cell Biol. 152, 997–1006.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 68, 850–858.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Cumming, R.C. (2008). Analysis of Global and Specific Changes in the Disulfide Proteome Using Redox 2D-PAGE. In: Hancock, J.T. (eds) Redox-Mediated Signal Transduction. Methods in Molecular Biology™, vol 476. Humana Press. https://doi.org/10.1007/978-1-59745-129-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-129-1_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-842-3
Online ISBN: 978-1-59745-129-1
eBook Packages: Springer Protocols