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

Cochemé, H.M. et al. (2012) Using the mitochondria-targeted ratiometric mass spectrometry probe MitoB to measure H₂O₂ in living Drosophila. Nature Protocols, 7(5), pp. 946-958. (doi: 10.1038/nprot.2012.035) (PMID:22517261)

Abstract

The role of hydrogen peroxide (H₂O₂) in mitochondrial oxidative damage and redox signaling is poorly understood, because it is difficult to measure H₂O₂ in vivo. Here we describe a method for assessing changes in H₂O₂ within the mitochondrial matrix of living <i>Drosophila</i>. 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 H₂O₂ 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 H₂O₂. 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 H₂O₂ in fly models of pathology and metabolic alteration, and it can also be extended to assess mitochondrial H₂O₂ production in mouse and cell culture studies.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Hartley, Professor Richard
• Authors: Cochemé, H.M., Logan, A., Prime, T.A., Abakumova, I., Quin, C., McQuaker, S., Patel, J.V., Fearnley, I.M., James, A.M., Porteous, C.M., Smith, R.A.J., Hartley, R.C., Partridge, L., and Murphy, M.P.
• College/School: College of Science and Engineering > School of Chemistry
• Journal Name: Nature Protocols
• Publisher: Nature Publishing
• ISSN: 1754-2189
• ISSN (Online): 1750-2799
• Published Online: 19 April 2012