title: Oxidative modifications of mitochondria complex II.
abstract: Increased superoxide (O2 (·-)) and nitric oxide (NO) production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In the complex II, oxidative impairment, decreased protein S-glutathionylation, and increased protein tyrosine nitration at the 70 kDa subunit occur in the post-ischemic myocardium (Zhang et al., Biochemistry 49:2529-2539, 2010; Chen et al., J Biol Chem 283:27991-28003, 2008; Chen et al., J Biol Chem 282: 32640-32654, 2007). To gain the deeper insights into ROS-mediated oxidative modifications relevant in myocardial infarction, isolated complex II is subjected to in vitro oxidative modifications with GSSG (to induce cysteine S-glutathionylation) or OONO(-) (to induce tyrosine nitration). Here, we describe the protocol to characterize the specific oxidative modifications at the 70 kDa subunit by nano-LC/MS/MS analysis. We further demonstrate the cellular oxidative modification with protein nitration/S-glutathionylation with immunofluorescence microscopy using the antibodies against 3-nitrotyrosine/glutathione and complex II 70 kDa polypeptide (AbGSC90) in myocytes under conditions of oxidative stress.
authors.affiliation: Proteomics and Mass Spectrometry Facility, Campus Chemical Instrument Center, The Ohio State University, Columbus, OH, USA.
authors.forename: Liwen
authors.initials: L
authors.lastname: Zhang
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Kang
authors.forename: Chwen-Lih
authors.initials: CL
authors.lastname: Chen
authors.forename: Kari B
authors.initials: KB
authors.lastname: Green
authors.forename: Yeong-Renn
authors.initials: YR
authors.lastname: Chen
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D042963:Electron Transport Complex II / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009203:Myocardial Infarction / Q000378:metabolism* / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009569:Nitric Oxide / Q000096:biosynthesis; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000494:pharmacology; D051381:Rats; D053719:Tandem Mass Spectrometry
chemical_list: D024101:Mitochondrial Proteins; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D042963:Electron Transport Complex II; D019803:Glutathione Disulfide