EPR Spin-Trapping and Nano LC MS/MS Techniques for DEPMPO/OOH and Immunospin-Trapping with Anti-DMPO

Mitochondrial superoxide (O2 •– ) production is an important mediator of oxidative cellular injury and pathogenesis of many diseases such as myocardial ischemia/reperfusion. The O2 •– generated in mitochondria acts as a redox signal triggering cellular events including apoptosis, proliferation, and senescence. The molecular mechanism of O2 •– 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 O2 •– production in mitochondria. O2 •– 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 O2 •– attack.