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.
Highlights
Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps
In addition to the different analytical approaches, lipidomics data published so far originated from very different mitochondria isolation methods: Either DC20–23 or UC on density gradients like Iodixanol[29], Ficoll and Sucrose[11,27], or Percoll[21,23,24]
This so-called MACS procedure has higher yields[26] and liver mitochondria isolated by MACS have been shown to exhibit higher oxygen consumption rates than mitochondria isolated by DC25
Summary
Subcellular lipidomics is a novel field of research that requires the careful combination of several pre-analytical and analytical steps. 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. We aimed to establish a valid, robust and sensitive workflow for the isolation of mitochondria from cell culture samples and subsequent lipid profiling by ultra performance liquid chromatography (UPLC)-LTQ-Orbitrap-mass spectrometry (MS). To this end, we systematically compared three different isolation procedures and the composition and characteristics of the resulting lipid profiles, focussing on the purity of the yielded mitochondrial fraction. Using liver-derived HepG2 cells, we defined a strategy for the valid, comprehensive and reproducible investigation of the lipidome of pure mitochondria based on as little as 100 μ g (by protein) of isolated sample material
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