Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila.

TitleUsing the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H2O2 in living Drosophila.
Publication TypeJournal Article
Year of Publication2012
AuthorsCochemé, HM, Logan, A, Prime, TA, Abakumova, I, Quin, C, McQuaker, SJ, Patel, JV, Fearnley, IM, James, AM, Porteous, CM, Smith, RAJ, Hartley, RC, Partridge, L, Murphy, MP
JournalNat Protoc
Volume7
Issue5
Pagination946-58
Date Published2012 Apr 19
ISSN1750-2799
KeywordsAnimals, Cells, Cultured, Chromatography, High Pressure Liquid, Drosophila, Hydrogen Peroxide, Mice, Mitochondria, Organophosphorus Compounds, Tandem Mass Spectrometry
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.

DOI10.1038/nprot.2012.035
Alternate JournalNat Protoc
Citation Key10.1038/nprot.2012.035
PubMed ID22517261
Grant ListBB/D020786/1 / / Biotechnology and Biological Sciences Research Council / United Kingdom
MC_U105663142 / / Medical Research Council / United Kingdom
/ / Wellcome Trust / United Kingdom