EPR spin-trapping and nano LC MS/MS techniques for DEPMPO/OOH and immunospin-trapping with anti-DMPO antibody in...

Mitochondrial superoxide (O(2) (-)) production is an important mediator of oxidative cellular injury and pathogenesis of many diseases such as myocardial ischemia/reperfusion. The O(2) (-) generated in mitochondria acts as a redox signal triggering cellular events including apoptosis, proliferation, and senescence. The molecular mechanism of O(2) (-) produced by electron transport chain components isolated from the inner membrane is investigated by the technique of EPR spin trapping with 5-diethoxylphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO), indicating that FMN/FMN-binding domain (complex I), ubiquinone (complex I and III), FAD/FAD-binding domain (complex II), and cytochrome b (complex III) control the mediation of O(2) (-) production in mitochondria. O(2) (-) generation by ETC also induces oxidative damage with protein radical formation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry are used to define the specific site of oxidative damage, indicating cysteine-206 and tyrosine-177 of complex I/51 kDa FMN-binding subunit and cysteine-655 of complex II/70 kDa FAD-binding subunit are involved in specific protein radical formation caused by O(2) (-) attack.