Proteomic Analysis of Redox-Dependent Changes Using Cysteine-Labeling 2D DIGE

  • Hong-Lin Chan
  • John Sinclair
  • John F. TimmsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 854)


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 words

Thiol-reactive cyanine dyes Two-dimensional difference gel electrophoresis Redox proteomics Mass spectrometry 


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Institute of Bioinformatics and Structural BiologyNational Tsing Hua UniversityHsinchuTaiwan
  2. 2.Cancer Proteomics Laboratory, EGA Institute for Women’s HealthUniversity College LondonLondonUK

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