EPR spin-trapping and nano LC MS/MS techniques for DEPMPO/ •OOH and immunospin-trapping with anti-DMPO antibody in mitochondrial electron transfer system

Abstract

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. © 2009 Humana Press.