Abstract
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 H 2 O 2 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. © 2012 Springer Science+Business Media, LLC.