Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics

摘要

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the “gold-standard” Folch or Bligh and Dyer recipes. Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the “gold-standard” Folch or Bligh and Dyer recipes. Recent developments in mass spectrometric technology enabled the comprehensive characterization of eukaryotic lipidomes, fostering the molecular biology of lipids and metabolism-related disorders (reviewed in Refs. 1.Han X. Gross R.W. Shotgun lipidomics: multidimensional MS analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Crossref PubMed Scopus (215) Google Scholar, 2.Wenk M.R. The emerging field of lipidomics.Nat. Rev. Drug Discov. 2005; 4: 594-610Crossref PubMed Scopus (1001) Google Scholar, 3.Piomelli D. Astarita G. Rapaka R. A neuroscientist's guide to lipidomics.Nat. Rev. Neurosci. 2007; 8: 743-754Crossref PubMed Scopus (274) Google Scholar, 4.van Meer G. Cellular lipidomics.EMBO J. 2005; 24: 3159-3165Crossref PubMed Scopus (411) Google Scholar). Typically, lipidome profiling by mass spectrometry proceeds along LC-MS or shotgun approaches. The former identifies and quantifies lipid species preseparated by normal or reversed-phase chromatography coupled online to a mass spectrometer, which is capable of fast acquisition of MS or MS/MS spectra (5.Yetukuri L. Katajamaa M. Medina-Gomez G. Seppanen-Laakso T. Vidal-Puig A. Oresic M. Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis.BMC Syst Biol. 2007; 1: 12-26Crossref PubMed Scopus (193) Google Scholar, 6.Sommer U. Herscovitz H. Welty F.K. Costello C.E. LC-MS-based method for the qualitative and quantitative analysis of complex lipid mixtures.J. Lipid Res. 2006; 47: 804-814Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 7.Hermansson M. Uphoff A. Kakela R. Somerharju P. Automated quantitative analysis of complex lipidomes by liquid chromatography/mass spectrometry.Anal. Chem. 2005; 77: 2166-2175Crossref PubMed Scopus (169) Google Scholar, 8.Haimi P. Uphoff A. Hermansson M. Somerharju P. Software tools for analysis of mass spectrometric lipidome data.Anal. Chem. 2006; 78: 8324-8331Crossref PubMed Scopus (167) Google Scholar). In contrast, in shotgun lipidomics, total lipid extracts are infused directly into a mass spectrometer, and the molecular characterization of lipid species relies either on the accurately determined m/z of precursor ions (9.Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring