Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling.

TitleDetection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling.
Publication TypeJournal Article
Year of Publication2007
AuthorsHurd, TR, Prime, TA, Harbour, ME, Lilley, KS, Murphy, MP
JournalJ Biol Chem
Volume282
Issue30
Pagination22040-51
Date Published2007 Jul 27
ISSN0021-9258
KeywordsAnimals, Electrophoresis, Electrophoresis, Gel, Two-Dimensional, Glutathione, Glutathione Disulfide, Hydrogen Peroxide, Mitochondria, Heart, Oxidation-Reduction, Proteins, Rats, Reactive Oxygen Species, Signal Transduction, Sulfhydryl Compounds
Abstract

Reactive oxygen species (ROS) produced by the mitochondrial 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 mitochondrial proteins involved in fatty acid and carbohydrate metabolism. 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 electrophoresis, 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 detection of thiol redox modifications by changes in the relative fluorescence 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 respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.

DOI10.1074/jbc.M703591200
Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.M703591200
PubMed ID17525152
Grant ListMC_U105663142 / / Medical Research Council / United Kingdom
MC_U105663148 / / Medical Research Council / United Kingdom