Chapter 19 Measuring redox changes to mitochondrial protein thiols with redox difference gel electrophoresis (redox-DIGE).

Low levels of reactive oxygen and nitrogen species (ROS and RNS) produced by the mitochondrial respiratory chain and ROS and RNS from other sources act as redox signals by oxidizing thiols on specific proteins. Because these thiol modifications occur on a relatively small number of proteins in the absence of bulk thiol changes, it is necessary to use sensitive methods to discover them. Recently, a number of methods have been developed to help facilitate the identification and characterization of redox-sensitive thiol proteins. In this chapter we describe one such method, redox difference gel electrophoresis (redox-DIGE), in which oxidized thiol proteins in redox-challenged samples are labeled with a thiol-reactive fluorescent tag and compared with those in control samples labeled with a different tag on the same 2-D gel. This enables the sensitive detection of redox-sensitive thiol proteins by measuring changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. With this method we have been able to identify several mitochondrial proteins whose thiol state and activity are altered by low levels of ROS from the respiratory chain, which may be an important and unexplored mode of mitochondrial redox signaling. Importantly, this method is not only applicable to studies in isolated mitochondria but can also be applied to more complicated systems such as intact cells and perhaps even whole organisms.