Detection of Reactive Oxygen Species-sensitive Thiol Proteins by Redox Difference Gel Electrophoresis

Abstract

Reactive oxygen species (ROS) produced by the mitochon-drial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochon-drial proteins involved in fatty acid and carbohydrate metabo-lism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel elec-trophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detec-tion of thiol redox modifications by changes in the relative flu-orescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the res-piratory chain may be an important and unexplored mode of mitochondrial redox signaling. Reactive oxygen and nitrogen species (ROS 2 and RNS) are used by mammalian cells as signaling molecules for redox reg-ulation (1, 2). Examples are hydrogen peroxide (H 2 O 2) and nitric oxide (NO), which are weak oxidants that can cross mem-branes and react with defined targets (2, 3). Modifications to protein thiols by ROS/RNS are an important aspect of redox signal transduction (2, 4, 5). These thiol modifications include oxidation to sulfenic acids, intra-and intermolecular disulfides, glutathionylation, sulfenyl-amide linkages, and S-nitrosation (6 –9). As many of these modifications can be reversed through interaction with glutathione or thioredoxin, protein thiol redox state can respond to the redox environment (10, 11). The mito-chondrial respiratory chain is the major source of ROS in most cells (12, 13) and is involved in redox signaling (14). The prox-imity of mitochondrial protein thiols to the major ROS source and the high matrix pH (ϳ8.0) make modification of mitochon-drial protein thiols by ROS more likely than for cytosolic thiols (15). Some mitochondrial proteins have shown ROS-depend-ent thiol oxidation:peroxiredoxin III (PrxIII) (16), NADP