Summary
A state of oxidative stress (OS) can occur when there is an imbalance between the rate of reactive oxygen species (ROS) production and their detoxification. Under OS conditions sulphur-containing residues are particularly susceptible to oxidation, and this can result in transient formation of intra- or inter-chain disulphide bridges. Diagonal electrophoresis is a relatively simple technique to analyse the formation of these bridges by sequential non-reducing/reducing electrophoresis. Proteins that do not form disulphides, electrophorese identically in both dimensions and form a diagonal after the second dimension, proteins that contained intra-chain disulphides lie above this diagonal, while those that formed inter-disulphides fall below the diagonal. These spots can be excised, tryptic digested, and identified by mass spectrometry. Identification of those proteins, which are reversibly modified, could play an important part in coupling redox status to protein function.
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
Leichert, L. and Jakob, U. (2006). Global methods to monitor the thiol-disulfide state of proteins in vivo. Antioxid. Redox Signal. 8, 763–772.
O’ Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Sommer, A. and Traut, R. R. (1974). Diagonal polyacyrlamide-dodecyl sulphate gel electrophoresis for the identification of ribosomal proteins crosslinked with emtyl-4-mercapto-butyrimidate. Proc. Natl. Acad. Sci. U.S.A. 71, 3946–3950.
Pe’er, I., Felder, C. E., Man, O., Silman, I., Sussman, J. L., and Beckmann, J. S. (2004). Proteomic signatures: amino acid and oligopeptide compositions differentiate among phyla. Proteins 54, 20–40.
Brennan, J. P., Wait, R., Begum, S., Bell, J. R., Dunn, M. J., and Eaton, P. (2004). Detection and mapping of widespread internal protein disulfide formation during oxidative stress using proteomics with diagonal electrophoresis. J. Biol. Chem. 279, 41352–42360.
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.
McDonagh, B. and Sheehan, D. (2007). Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: Proteomic identification of target proteins. Proteomics, 7, 3395–3403.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227,680–684.
Acknowledgments
I acknowledge an Embark fellowship from the Irish Research Council for Science, Engineering and technology.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
McDonagh, B. (2009). Diagonal Electrophoresis for Detection of Protein Disulphide Bridges. 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_19
Download citation
DOI: https://doi.org/10.1007/978-1-59745-281-6_19
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-937-6
Online ISBN: 978-1-59745-281-6
eBook Packages: Springer Protocols