A fluorescent labelling technique to detect changes in the thiol redox state of proteins following mild oxidative stress /

There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The "2-tag fluorescent labelling technique" was able to label both reduced and oxidized cysteine residues of thiol proteins to arrive at a redox ratio which was comparable across different samples. The redox ratio was used to compare samples of untreated Jurkats against mild oxidatively stressed cells as well as low oxygen treated cells. Proteins showing evidence of thiol modification were identified through two dimensional electrophoresis and mass spectrometry. A variety of proteins were found to have modified thiols as a result of either a mild oxidative stress treatment or low oxygen treatment. Several proteins that were previously known to respond to changes in oxidative stress were identified. Additionally, proteins not previously linked to oxidative stress were identified. This data indicates that the method can successfully detect proteins responding to mild changes in oxidizing conditions.