title: Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila.
abstract: The role of hydrogen peroxide (H(2)O(2)) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H(2)O(2) in vivo. Here we describe a method for assessing changes in H(2)O(2) within the mitochondrial matrix of living Drosophila. We use a ratiometric mass spectrometry probe, MitoB ((3-hydroxybenzyl)triphenylphosphonium bromide), which contains a triphenylphosphonium cation component that drives its accumulation within mitochondria. The arylboronic moiety of MitoB reacts with H(2)O(2) to form a phenol product, MitoP. On injection into the fly, MitoB is rapidly taken up by mitochondria and the extent of its conversion to MitoP enables the quantification of H(2)O(2). To assess MitoB conversion to MitoP, the compounds are extracted and the MitoP/MitoB ratio is quantified by liquid chromatography-tandem mass spectrometry relative to deuterated internal standards. This method facilitates the investigation of mitochondrial H(2)O(2) in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H(2)O(2) production in mouse and cell culture studies.
authors.affiliation: Medical Research Council, Mitochondrial Biology Unit, Cambridge, UK.
authors.forename: Helena M
authors.initials: HM
authors.lastname: Cochemé
authors.forename: Angela
authors.initials: A
authors.lastname: Logan
authors.forename: Tracy A
authors.initials: TA
authors.lastname: Prime
authors.forename: Irina
authors.initials: I
authors.lastname: Abakumova
authors.forename: Caroline
authors.initials: C
authors.lastname: Quin
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: McQuaker
authors.forename: Jigna V
authors.initials: JV
authors.lastname: Patel
authors.forename: Ian M
authors.initials: IM
authors.lastname: Fearnley
authors.forename: Andrew M
authors.initials: AM
authors.lastname: James
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Porteous
authors.forename: Robin A J
authors.initials: RA
authors.lastname: Smith
authors.forename: Richard C
authors.initials: RC
authors.lastname: Hartley
authors.forename: Linda
authors.initials: L
authors.lastname: Partridge
authors.forename: Michael P
authors.initials: MP
authors.lastname: Murphy
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004330:Drosophila / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: C558697:MItoB compound; D009943:Organophosphorus Compounds; D006861:Hydrogen Peroxide