title: Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry.
abstract: The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo.
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: Logan
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK.
authors.forename: Victoria R
authors.initials: VR
authors.lastname: Pell
authors.affiliation: Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Shaffer
authors.affiliation: Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Cameron
authors.initials: C
authors.lastname: Evans
authors.affiliation: Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Stanley
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Ellen L
authors.initials: EL
authors.lastname: Robb
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Tracy A
authors.initials: TA
authors.lastname: Prime
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK; Dana-Farber Cancer Institute and Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA; Department of Cell Biology, Harvard University Medical School, Boston, MA 02115, USA.
authors.forename: Edward T
authors.initials: ET
authors.lastname: Chouchani
authors.affiliation: Institute of Healthy Ageing, Department of Genetics, Evolution, and Environment, University College London, Gower Street, London WC1E 6BT, UK; MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.
authors.forename: Helena M
authors.initials: HM
authors.lastname: Cochemé
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Ian M
authors.initials: IM
authors.lastname: Fearnley
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Vidoni
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: James
authors.affiliation: Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand; Department Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Porteous
authors.affiliation: Institute of Healthy Ageing, Department of Genetics, Evolution, and Environment, University College London, Gower Street, London WC1E 6BT, UK.
authors.forename: Linda
authors.initials: L
authors.lastname: Partridge
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Krieg
authors.affiliation: Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
authors.forename: Robin A J
authors.initials: RA
authors.lastname: Smith
authors.affiliation: Medical Research Council Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK. Electronic address: mpm@mrc-mbu.cam.ac.uk.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Murphy
mesh_terms: D000818:Animals; D002460:Cell Line; D057930:Click Chemistry / Q000379:methods*; D053078:Membrane Potential, Mitochondrial; D008810:Mice, Inbred C57BL; D015335:Molecular Probes / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015335:Molecular Probes