Summary
Contamination from subcellular organelles and myelin has hindered attempts to characterize the lipidome of brain mitochondria. A high degree of mitochondrial purity is required for accurate measurements of the content and molecular species composition of mitochondrial lipids. We devised a discontinuous Ficoll and sucrose gradient procedure for the isolation and purification of brain mitochondria free from any detectable contamination. Shotgun lipidomics was used to analyze the lipid composition of the brain mitochondria. These procedures can be used to determine whether intrinsic lipid abnormalities underlie mitochondrial dysfunction associated with neurological and neurodegenerative diseases.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kiebish M.A., Han X., Cheng H., Lunceford A., Clarke C.F., Moon H., Chuang J.H. and Seyfried T.N. (2008) Lipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria. J Neurochem 106, 299–312.
Lai J.C., Walsh J.M., Dennis S.C. and Clark J.B. (1977) Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J Neurochem 28, 625–631.
Brown M.R., Sullivan P.G. and Geddes J.W. (2006) Synaptic mitochondria are more susceptible to Ca2+overload than nonsynaptic mitochondria. J Biol Chem 281, 11658–11668.
Dagani F., Gorini A., Polgatti M., Villa R.F. and Benzi G. (1983) Synaptic and non-synaptic mitochondria from rat cerebral cortex. Characterization and effect of pharmacological treatment on some enzyme activities related to energy transduction. Farmaco [Sci] 38, 584–594.
Villa R.F., Gorini A., Geroldi D., Lo Faro A. and Dell’Orbo C. (1989) Enzyme activities in perikaryal and synaptic mitochondrial fractions from rat hippocampus during development. Mech Ageing Dev 49, 211–225.
Wallace D.C. (2001) A mitochondrial paradigm for degenerative diseases and ageing. Novartis Found Symp 235, 247–263; discussion 263–266.
Daum G. (1985) Lipids of mitochondria. Biochim Biophys Acta 822, 1–42.
Hoch F.L. (1992) Cardiolipins and biomembrane function. Biochim Biophys Acta 1113, 71–133.
Stuart J.A., Gillis T.E. and Ballantyne J.S. (1998) Remodeling of phospholipid fatty acids in mitochondrial membranes of estivating snails. Lipids 33, 787–793.
Rostovtseva T.K. and Bezrukov S.M. (2008) VDAC regulation: role of cytosolic proteins and mitochondrial lipids. J Bioenerg Biomembr 40, 163–170.
Campbell A.M. and Chan S.H. (2008) Mitochondrial membrane cholesterol, the voltage dependent anion channel (VDAC), and the Warburg effect. J Bioenerg Biomembr 40, 193–197.
Han X. and Gross R.W. (2005) Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass spectrometry reviews 24, 367–412.
Yang K., Zhao Z., Gross R.W. and Han X. (2007) Shotgun lipidomics identifies a paired rule for the presence of isomeric ether phospholipid molecular species. PLoS ONE 2, e1368.
Han X. and Gross R.W. (2005) Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes. Expert review of proteomics 2, 253–264.
Han X., Holtzman D.M. and McKeel D.W., Jr. (2001) Plasmalogen deficiency in early Alzheimer’s disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry. J Neurochem 77, 1168–1180.
Cheng H., Mancuso D.J., Jiang X., Guan S., Yang J., Yang K., Sun G., Gross R.W. and Han X. (2008) Shotgun lipidomics reveals the temporally dependent, highly diversified cardiolipin profile in the mammalian brain: temporally coordinated postnatal diversification of cardiolipin molecular species with neuronal remodeling. Biochemistry 47, 5869–5880.
Han X., Yang K., Yang J., Cheng H. and Gross R.W. (2006) Shotgun lipidomics of cardiolipin molecular species in lipid extracts of biological samples. J Lipid Res 47, 864–879.
Zischka H., Lichtmannegger J., Jagemann N., Jennen L., Hamoller D., Huber E., Walch A., Summer K.H. and Gottlicher M. (2008) Isolation of highly pure rat liver mitochondria with the aid of zone-electrophoresis in a free flow device (ZE-FFE). Methods Mol Biol 424, 333–348.
Sims N.R. (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55, 698–707.
Sims N.R. and Anderson M.F. (2008) Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 3, 1228–1239.
Dagani F., Zanada F., Marzatico F. and Benzi G. (1985) Free mitochondria and synaptosomes from single rat forebrain. A comparison between two known subfractionation techniques. J Neurochem 45, 653–656.
Taylor S.W., Warnock D.E., Glenn G.M., Zhang B., Fahy E., Gaucher S.P., Capaldi R.A., Gibson B.W. and Ghosh S.S. (2002) An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria. J Proteome Res 1, 451–458.
Stocco D.M. and Hutson J.C. (1980) Characteristics of mitochondria isolated by rate zonal centrifugation from normal liver and Novikoff hepatomas. Cancer Res 40, 1486–1492.
Graham J.M. (2001) Purification of a crude mitochondrial fraction by density-gradient centrifugation. Curr Protoc Cell Biol Chapter 3, Unit 3 4.
Lai J.C. and Clark J.B. (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem J 154, 423–432.
Mena E.E., Hoeser C.A. and Moore B.W. (1980) An improved method of preparing rat brain synaptic membranes. Elimination of a contaminating membrane containing 2´,3´-cyclic nucleotide 3´-phosphohydrolase activity. Brain Res 188, 207–s31.
Rendon A. and Masmoudi A. (1985) Purification of non-synaptic and synaptic mitochondria and plasma membranes from rat brain by a rapid Percoll gradient procedure. J Neurosci Methods 14, 41–51.
Battino M., Bertoli E., Formiggini G., Sassi S., Gorini A., Villa R.F. and Lenaz G. (1991) Structural and functional aspects of the respiratory chain of synaptic and nonsynaptic mitochondria derived from selected brain regions. J Bioenerg Biomembr 23, 345–363.
Cheng H., Guan S. and Han X. (2006) Abundance of triacylglycerols in ganglia and their depletion in diabetic mice: implications for the role of altered triacylglycerols in diabetic neuropathy. J Neurochem 97, 1288–300.
Han X., Yang J., Cheng H., Ye H. and Gross R.W. (2004) Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry. Anal Biochem 330, 317–331.
Brigande J.V., Platt F.M. and Seyfried T.N. (1998) Inhibition of glycosphingolipid biosynthesis does not impair growth or morphogenesis of the postimplantation mouse embryo. J Neurochem 70, 871–882.
Petrozzi L., Ricci G., Giglioli N.J., Siciliano G. and Mancuso M. (2007) Mitochondria and neurodegeneration. Biosci Rep 27, 87–104.
Beal M.F. (2005) Mitochondria take center stage in aging and neurodegeneration. Ann Neurol 58, 495–505.
Calabrese V., Scapagnini G., Giuffrida Stella A.M., Bates T.E. and Clark J.B. (2001) Mitochondrial involvement in brain function and dysfunction: relevance to aging, neurodegenerative disorders and longevity. Neurochem Res 26, 739–64.
Bowling A.C. and Beal M.F. (1995) Bioenergetic and oxidative stress in neurodegenerative diseases. Life Sciences 56, 1151–1171.
Kiebish M.A., Han X., Cheng H., Chuang J.H. and Seyfried T.N. (2008) Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: Lipidomic evidence supporting the Warburg theory of cancer. J Lipid Res 49, 2545–2556
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kiebish, M.A., Han, X., Seyfried, T.N. (2009). Examination of the Brain Mitochondrial Lipidome Using Shotgun Lipidomics. In: Armstrong, D. (eds) Lipidomics. Methods in Molecular Biology, vol 579. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-322-0_1
Download citation
DOI: https://doi.org/10.1007/978-1-60761-322-0_1
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-321-3
Online ISBN: 978-1-60761-322-0
eBook Packages: Springer Protocols