title: Purity matters: A workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples.
abstract: 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.
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Department of Diagnostic Laboratory Medicine, University Hospital Tuebingen, Tuebingen, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Kappler
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Department of Diagnostic Laboratory Medicine, University Hospital Tuebingen, Tuebingen, Germany.|Department of Molecular Diabetology, Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Centre Munich at the University of Tuebingen, Tuebingen, Germany.|German Center for Diabetes Research (DZD), Tuebingen, Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Häring
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Department of Diagnostic Laboratory Medicine, University Hospital Tuebingen, Tuebingen, Germany.|Department of Molecular Diabetology, Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Centre Munich at the University of Tuebingen, Tuebingen, Germany.|German Center for Diabetes Research (DZD), Tuebingen, Germany.
authors.forename: Cora
authors.initials: C
authors.lastname: Weigert
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Department of Diagnostic Laboratory Medicine, University Hospital Tuebingen, Tuebingen, Germany.|Department of Molecular Diabetology, Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Centre Munich at the University of Tuebingen, Tuebingen, Germany.|German Center for Diabetes Research (DZD), Tuebingen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Lehmann
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
authors.forename: Guowang
authors.initials: G
authors.lastname: Xu
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Department of Diagnostic Laboratory Medicine, University Hospital Tuebingen, Tuebingen, Germany.
authors.forename: Miriam
authors.initials: M
authors.lastname: Hoene
mesh_terms: D002308:Cardiolipins / Q000302:isolation & purification / Q000378:metabolism; D018929:Cell Culture Techniques; D002498:Centrifugation; D016000:Cluster Analysis; D056945:Hep G2 Cells; D006801:Humans; D018189:Immunomagnetic Separation; D050356:Lipid Metabolism; D008055:Lipids / Q000302:isolation & purification*; D055432:Metabolomics / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism; D057188:Workflow
chemical_list: D002308:Cardiolipins; D008055:Lipids