An Improved High-Throughput Lipid Extraction Method for the Analysis of Human Brain Lipids
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We have developed a protocol suitable for high-throughput lipidomic analysis of human brain samples. The traditional Folch extraction (using chloroform and glass–glass homogenization) was compared to a high-throughput method combining methyl-tert-butyl ether (MTBE) extraction with mechanical homogenization utilizing ceramic beads. This high-throughput method significantly reduced sample handling time and increased efficiency compared to glass–glass homogenizing. Furthermore, replacing chloroform with MTBE is safer (less carcinogenic/toxic), with lipids dissolving in the upper phase, allowing for easier pipetting and the potential for automation (i.e., robotics). Both methods were applied to the analysis of human occipital cortex. Lipid species (including ceramides, sphingomyelins, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserines) were analyzed via electrospray ionization mass spectrometry and sterol species were analyzed using gas chromatography mass spectrometry. No differences in lipid species composition were evident when the lipid extraction protocols were compared, indicating that MTBE extraction with mechanical bead homogenization provides an improved method for the lipidomic profiling of human brain tissue.
KeywordsLipidomics Human-brain Ceramide Sphingomyelin Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Sterol
Coefficient of variation
Collision cell exit potential
Electrospray ionization mass spectrometry
Gas chromatography mass spectrometry
Multiple reaction monitoring
Adult brain tissue was received from the Sydney Brain Bank at Neuroscience Research Australia which is supported by the National Health and Medical Research Council of Australia, the University of New South Wales and Neuroscience Research Australia. This research was supported by a project grant (APP1008307) from the Australian National Health and Medical Research Council (NHMRC) awarded to GMH and BG. TWM and BG are supported by Australian Research Council Future Fellowships (FT110100249 and FT0991986, respectively). GMH is supported by an NHMRC Principal Research Fellowship (630434).
Conflict of interest
All authors disclose that there are no conflicts of interest.
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