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Kappler, L.* ; Li, J.* ; Häring, H.-U. ; Weigert, C. ; Lehmann, R. ; Xu, G.* ; Hoene, M.*

Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples.

Sci. Rep. 6:21107 (2016)

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Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. To define a reliable strategy for mitochondrial lipid profiling, we performed a systematic comparison of different mitochondria isolation procedures by western blot analyses and comprehensive high-resolution lipidomics. Using liver-derived HepG2 cells, we compared three common mitochondria isolation methods, differential centrifugation (DC), ultracentrifugation (UC) and a magnetic bead-assisted method (MACS). In total, 397 lipid species, including 32 cardiolipins, could be quantified in only 100 μg (by protein) of purified mitochondria. Mitochondria isolated by UC showed the highest enrichment in the mitochondria-specific cardiolipins as well as their precursors, phosphatidylglycerols. Mitochondrial fractions obtained by the commonly used DC and the more recent MACS method contained substantial contaminations by other organelles. Employing these isolation methods when performing lipidomics analyses from cell culture mitochondria may lead to inaccurate results. To conclude, we present a protocol how to obtain reliable mitochondria-specific lipid profiles from cell culture samples and show that quality controls are indispensable when performing mitochondria lipidomics.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Functional Mitochondria; Brain Mitochondria; Mass-spectrometry; Skeletal-muscle; Rat-kidney; Liver; Cardiolipin; Organelle; Disease; Health

ISSN (print) / ISBN 2045-2322

e-ISSN 2045-2322

Journal Scientific Reports

Quellenangaben Volume: 6, Article Number: 21107

Publisher Nature Publishing Group

Publishing Place London

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Diabetes Research and Metabolic Diseases (IDM)