Proteomic Analysis of Redox-Dependent Changes Using Cysteine-Labeling 2D DIGE
Redox-modification of proteins plays an important role in the regulation of protein function and cellular physiology and in pathological conditions such as oncogenic activation, inhibition of tumor suppression, and ischemia reperfusion injury. This occurs, at least in part, through the reduction or oxidation of cysteine groups in these proteins resulting in the modulation of their activities. Herein, we focus on the development of a pair of cysteine-labeling iodoacetylated cyanine dyes (ICy3/5) for two-dimensional difference gel electrophoresis (2D DIGE) to monitor redox-dependent changes on cysteine residues. The method is applied to a cellular model of human mammary luminal epithelial cells treated with H2O2 to induce oxidative stress. Differences in labeling are caused either by differential protein expression or from the loss or gain of reactive thiol groups of cysteines in response to oxidative stress. Proteins displaying differential labeling would then be picked for MS-based identification. In summary, this cysteine-labeling 2D-DIGE approach provides an MS-compatible and reproducible technique for identifying alterations in the expression and redox-modification of free thiol-containing proteins.
Key wordsThiol-reactive cyanine dyes Two-dimensional difference gel electrophoresis Redox proteomics Mass spectrometry
- 7.Lai, T. C., Chou, H. C., Chen, Y. W., Lee, T. R., Chan, H. T., Shen, H. H., Lee, W. T., Lin, S. T., Lu, Y. C., Wu, C. L., and Chan, H. L. (2010) Secretomic and Proteomic Analysis of Potential Breast Cancer Markers by Two-Dimensional Differential Gel Electrophoresis, J Proteome Res 9, 1302–1322.Google Scholar
- 11.Lee, J. S., Ma, Y. B., Choi, K. S., Park, S. Y., Baek, S. H., Park, Y. M., Zu, K., Zhang, H., Ip, C., Kim, Y. H., and Park, E. M. (2006) Neural network-based analysis of thiol proteomics data in identifying potential selenium targets, Preparative biochemistry & biotechnology 36, 37–64.CrossRefGoogle Scholar
- 13.Chan, H. L., Gharbi, S., Gaffney, P. R., Cramer, R., Waterfield, M. D., and Timms, J. F. (2005) Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis, Proteomics 5, 2908–2926.PubMedCrossRefGoogle Scholar
- 14.Chan, H. L., Gaffney, P. R., Waterfield, M. D., Anderle, H., Peter Matthiessen, H., Schwarz, H. P., Turecek, P. L., and Timms, J. F. (2006) Proteomic analysis of UVC irradiation-induced damage of plasma proteins: Serum amyloid P component as a major target of photolysis, FEBS Lett 580, 3229–3236.PubMedCrossRefGoogle Scholar
- 17.Bruschi, M., Grilli, S., Candiano, G., Fabbroni, S., Della Ciana, L., Petretto, A., Santucci, L., Urbani, A., Gusmano, R., Scolari, F., and Ghiggeri, G. M. (2009) New iodo-acetamido cyanines for labeling cysteine thiol residues. A strategy for evaluating plasma proteins and their oxido-redox status, Proteomics 9, 460–469.Google Scholar