We are very interested in discovering new redox regulated proteins. Therefore, we have developed Redox Proteomics techniques, which allow us to determine the exact thiol-disulfide state of hundreds of cellular proteins in a single experiment.
Redox-Proteomics allows us to see the global changes in the thiol-disulfide status of proteins that happen in the cell when it is exposed to oxidative stress conditions. In this way we can learn about the oxidative stress that occurs in aging organisms and how cells can better cope with it.
When we combine redox proteomics with genetic experiments, we can also use it to find substrate proteins of oxidoreductases.
We can make a visual “snapshot” of the redox state of the cell with 2D gels. In this snapshot, oxidized proteins are visible as red spots, while reduced or cysteine free proteins appear as green spots.
Here we compare wild type E. coli (panel A) to a knock-out mutant in the main periplasmic thiol-oxidase DsbA (panel B). All the red spots on the wild type gel that turned yellowish-green on the mutant gel need DsbA to form disulfide bonds.
If we want a more detailed view, we can use mass spectrometry. This allows us to pin down the redox sensitive cysteine in the amino acid sequence of a protein. Using a technique we call OxICAT, we can monitor the oxidation state by a tell-tale mass shift of 9 Dalton per oxidized cysteine.
Here you see the mass spectrum of a peptide of the redox sensitive glycolytic protein GapDH in E. coli. Its active site cysteine is almost 100 % oxidized and presumably forms a disulfide bond with another nearby cysteine when E. coli is exposed to the important physiological oxidative stressor NaOCl, also known as household bleach.