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

Abstract

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 T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (151) Google Scholar) or on the detection of specific fragment ions or neutral losses in tandem mass spectrometric experiments (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, 9.Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (151) Google Scholar, 10.Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nanoelectrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (736) Google Scholar, 11.Han X. Yang K. Yang J. Fikes K.N. Cheng H. Gross R.W. Factors influencing the electrospray intrasource separation and selective ionization of glycerophospholipids.J. Am. Soc. Mass Spectrom. 2006; 17: 264-274Crossref PubMed Scopus (93) Google Scholar, 12.Ejsing C.S. Duchoslav E. Sampaio J. Simons K. Bonner R. Thiele C. Ekroos K. Shevchenko A. Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning.Anal. Chem. 2006; 78: 6202-6214Crossref PubMed Scopus (331) Google Scholar). Regardless of the analytical approach used, its success depends on the completeness of the extraction of lipids from corresponding cells, fluids, or tissues. Lipids of all major classes could be recovered via chloroform/methanol extraction, typically according to the Folch, Lees, and Sloane Stanley (13.Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) or Bligh and Dyer (14.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar) recipes (15.Watson A.D. Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: a global approach to lipid analysis in biological systems.J. Lipid Res. 2006; 47: 2101-2111Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), in which they are mostly enriched in the chloroform phase. Electrospray mass spectrometry, a major tool for analyzing complex lipidomes, is particularly sensitive towards the quality of lipid extracts. Coextracted components of biological matrix and salts (often, without further definition, termed background) affect both the sensitivity and specificity of lipid analysis. Often, abundant background ions obscure lipid precursors, and their MS/MS spectra are densely populated with “ghost” peaks and abundant chemical noise. Adducts with common background cations (sodium, potassium) and anions (chloride) increase the ambiguity of molecular species assignment and affect the accuracy of quantitative determination. Because of the higher density of chloroform compared with a water/methanol mixture, it forms the lower phase of the two-phase partitioning system. While collecting the chloroform fraction, a glass pipette or a needle of the pipetting robot reaches it through a voluminous layer of nonextractable insoluble matrix, usually residing at the interface of the water/methanol and chloroform phases. However, even a minute amount of insoluble precipitate accidentally grabbed together with the chloroform fraction clogs the electrospray ion source or LC system, because of the micrometer size of the spraying orifice and/or connecting tubing. We note that, because of high density and the viscosity of chloroform, centrifugation is usually of little help. Although mass spectrometry enables lipid profiling at the low femtomole level, much higher amounts are usually required to circumvent the difficulties in handling microvolumes of total extracts and ensure the sufficient stability of the analytical pipeline. Additionally, the known carcinogenicity of chloroform involves considerable health risk for laboratory personnel (16.Nagano K. Kano H. Arito H. Yamamoto S. Matsushima T. Enhancement of renal carcinogenicity by combined inhalation and oral exposures to chloroform in male rats.J. Toxicol. Environ. Health A. 2006; 69: 1827-1842Crossref PubMed Scopus (22) Google Scholar). Also, chloroform decomposition yields phosgene and hydrochloric acid, inflicting chemical modification of labile lipid species (17.Schmid P. Hunter E. Calvert J. Extraction and purification of lipids. III. Serious limitations of chloroform and chloroform-methanol in lipid investigations.Physiol. Chem. Phys. Med. NMR. 1973; 5: 151-155Google Scholar). Here, we report an extraction protocol specifically developed for shotgun profiling of complex lipidomes from samples with excessive amounts of biological matrices. Lipid extraction by methyl-tert-butyl ether (MTBE)/methanol (18.Thomann W.R. Hill G.B. Modified extraction procedure for gas-liquid chromatography applied to the identification of anaerobic bacteria.J. Clin. Microbiol. 1986; 23: 392-394Crossref PubMed Google Scholar, 19.Kuyukina M.S. Ivshina I.B. Philp J.C. Christofi N. Dunbar S.A. Ritchkova M.I. Recovery of Rhodococcus biosurfactants using methyl tertiary-butyl ether extraction.J. Microbiol. Methods. 2001; 46: 149-156Crossref PubMed Scopus (135) Google Scholar) greatly simplifies sample handling and enables automated processing of minute amounts of biological samples. Rigorous testing established that the recovery of lipid species of almost all major classes is the same or better than was typically achieved by the Folch recipe (13.Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), which is generally regarded the “gold standard” in lipid biochemistry. Synthetic lipid standards were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL); MTBE and 2-propanol were from Sigma-Aldrich Chemie GmbH (Munich, Germany). Chloroform, methanol, and ammonium acetate were LC grade; water with 0.1% ammonium acetate was LC-MS grade and purchased from Fluka (Buchs, Switzerland). LC-MS-grade water was purchased from Fisher Scientific (Loughborough, UK). Escherichia coli (NA-22 strain) were grown on Luria-Bertani medium, collected by centrifugation, washed three times by M9 solution (22 mM KH2PO4, 22 mM Na2HPO4, 85 mM NaCl, and 1 mM MgSO4) followed by rinsing with 0.1% ammonium acetate, and frozen. A sample of mouse brain tissue was dissected from adult mouse of NMRI strain. Brain hemispheres were separated and minced into small pieces in ice-cold 0.1% ammonium acetate followed by homogenization in a Potter homogenizer. The daf-22 strain of Caenorhabditis elegans was grown on NGM agar plates with E. coli (NA-22 strain) as a food source (20.Brenner S. The genetics of Caenorhabditis elegans.Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). To collect eggs, worms were bleached with basic hypochlorite solution as described (21.Sulston J. Hodgkin J. Methods.in: Wood W.B. The Nematode Caenorhabditis elegans. Cold Spring Harbor Laboratory Press, New York1988: 587-606Google Scholar). To remove worm debris, egg suspension was filtered through 80 μm nylon mesh, rinsed with LC-MS-grade water, and frozen in liquid nitrogen in Mass spectrometric analysis was on a mass with a ion source was to to and the source was by lipid samples were in mM ammonium acetate in and infused at the of A sample of of electrospray acquisition experiments were as described D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). The analytical was the and were the m/z MS/MS was for the of Lipid species were and using D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). lipid extracts were analyzed by on a mass with an electrospray ion source as described G. B. J. G. quantification of and by electrospray ionization tandem mass spectrometry coupled with PubMed Scopus Google Scholar, G. M. R. T. B. G. quantification of and by electrospray ionization tandem mass spectrometry 2006; PubMed Scopus Google Scholar). of total lipid extracts was for lipid analysis using a and an Germany). A of mM ammonium acetate was through the at the of for followed by for and to for were for of and upon lipid and were analyzed by D. G. R. G. Shevchenko A. Shotgun lipidomics by tandem mass spectrometry acquisition 2007; PubMed Scopus Google Scholar). and was as described G. B. J. G. quantification of and by electrospray ionization tandem mass spectrometry coupled with PubMed Scopus Google Scholar, G. M. B. R. B. A. G. Quantitative of species from cellular extracts by electrospray ionization tandem mass spectrometry Lipid Res. Full Text Full Text PDF PubMed Google mass spectrometric or for species in the acquisition lipidomics approach (22) by of fragment ions in MS/MS for species in the acquisition lipidomics approach D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar) by of fragment ions Electrospray ionization tandem mass spectrometry of Am. Soc. Mass Spectrom. PubMed Scopus Google Scholar) in MS/MS in a analysis of lipid extracts was on plates were developed with an system, followed by Lipid were by spraying the plates with in and at was to a sample which was into a glass tube with a and the tube was of MTBE was and the was for 1 at in a separation was by of of at the sample was at for The upper phase was and the lower phase was with of the mixture, was to the of the upper phase by and collecting the upper organic were in a To sample of was to the organic phase of centrifugation. lipids were in of for was to of the sample and of was the was for 1 at in a and phase separation was by of The was for at and at for The lower phase was and the upper phase was washed with of the mixture, was to the of the lower phase organic were in a and in of for of of lipid in were into and in a A total of of water was to and lipid extraction was according to the Folch or MTBE protocol as described organic were and in 1 of of lipid of the same that as an and were samples were by pipetting the same of lipid into and in the same amount of the extraction was in and was analyzed three MS spectra were for with an of 1 The lipid recovery was as the of the of peaks of the and the of the same lipid In a of the lipid was as described of water of E. coli suspension was to the The recovery was by multiple in ion as the of the of the fragment with m/z from the and the from grown on were collected by centrifugation, washed with 0.1% ammonium acetate in water, and in 0.1% ammonium in were into glass three were according to Folch and the three were with MTBE as described Lipid extracts were with and analyzed Lipid were by the of by which specific neutral precursor ion and D. G. R. G. Shevchenko A. Shotgun lipidomics by tandem mass spectrometry acquisition 2007; PubMed Scopus Google Scholar). E. coli of and lipid classes were by neutral of with m/z and m/z The was determined in ion by precursor ion for the fragment with m/z of lipid species were to the of of all lipid species of the lipid Brain tissue from adult mouse NMRI strain was rinsed in 0.1% ammonium acetate in water, into small and in 0.1% ammonium acetate in water in a Potter on of of were in and according to the Folch and MTBE The lipid extracts were with and analyzed for and were as described of the suspension in MS water of C. elegans of or were to three of and three were according to the Folch protocol and three were according to the MTBE To lipid was with and analyzed in and lipid were determined as described Lipid extracts were from of into glass lipid extraction, standards solution and in chloroform were into the same and Additionally, samples were by the of known of lipid Bligh and Dyer extraction, of was to the of with of the was and for 1 at (14.Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43133) Google Scholar). separation was achieved by 1 of and 1 of MTBE extraction, 80 of water was to the samples and as described extraction of the lower phase was of organic phase collected according to both was further by a pipetting robot with To the needle was washed with both lipid extraction the organic phase was recovered at a from the lower chloroform and upper MTBE were to by the phase. of the was with for the chloroform phase to sample were to glass In of the organic phase was recovered from of which was for analysis and the was for analysis. The was removed by centrifugation, and lipids were in and of mM ammonium acetate for and To the recoveries of and three samples with medium, and high lipid by a determined total as medium, were in of lipid species were determined by of a sample times as described that MTBE could chloroform in systems for lipid The established extraction in similar to the Folch or Bligh and Dyer In the samples were a extraction and biological Lipids were recovered into the MTBE because of its lower density, was the upper phase of the two-phase system. In to the Folch nonextractable matrix in the phase at the bottom of the extraction the organic phase enriched with lipids was easily by the from the was for lipids from C. which voluminous nonextractable that the chloroform fraction collected with the Folch an was required to it analysis. The MTBE extraction procedure was in three we determined the recoveries of lipid standards of classes and compared with the recoveries achieved by the Folch we established that the recovery of both was almost we the Folch recipe as a to lipid yields were by biological and they on the of lipid MTBE extraction was automated and applied for of we determined lipid recoveries were using the Folch and MTBE recipes. To of lipid standards of lipid classes was and their recoveries were determined by mass spectrometry using standards of the same and similar mass and were known on the lipid the recovery achieved by both was The was the which was by The same was in the into E. coli total lipid was recovered by Folch and by we that, in further we could MTBE extraction to the Folch upon the of peaks of of lipid standards by MTBE and Folch extraction are of of lipid standards Folch or MTBE extraction were determined by with the of peaks of the corresponding in a are of of lipid standards Folch or MTBE extraction were determined by with the of peaks of the corresponding The MTBE and Folch were applied to lipids from as E. mouse and C. an of and animal the same samples were in by both recovered lipids were analyzed by and lipid species were by shotgun profiling on a mass D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). The E. coli lipidome mostly of and in lipid of Escherichia coli from with organic and with food Environ. Microbiol. PubMed Google Scholar). to species and survey mass spectra were that the extraction yields were similar for both and were of lipid and the of the was by analysis the m/z of background peaks were lipid profiling in ion the same total and of and species In we species of and species of which with D. C. N. S. V. Lipid of of Escherichia coli by liquid mass spectrometry using electrospray Mass Spectrom. 2007; PubMed Scopus Google Scholar). the Folch and MTBE were applied to lipids from adult mouse brain MS analysis of the extracts almost spectra and was further by analysis The profiling lipid species from lipid classes A total of of species lipids from the most abundant and classes the were to species from and the of major and species recovered by both was almost that corresponding organic and were with lipids. MTBE and Folch extraction of C. elegans of lipid in lipid species from major lipid classes of in ion species with of were in both extracts The MTBE protocol was further for automated extraction of and in lipidomics screens G. B. J. G. quantification of and by electrospray ionization tandem mass spectrometry coupled with PubMed Scopus Google Scholar, G. M. R. T. B. G. quantification of and by electrospray ionization tandem mass spectrometry 2006; PubMed Scopus Google Scholar). In the same samples were by the Bligh and Dyer method using the same The extraction recovery was by processing three samples with medium, and high lipid Bligh and Dyer and MTBE recovered the same amount of lipids of major classes with similar of which was MTBE extraction recovered from samples compared with the Bligh and Dyer of lipids extraction of samples according to the MTBE or Bligh and Dyer of lipid recovered automated MTBE or Bligh and Dyer The are of experiments in to the total of the samples medium, determined by Lipid to the total of the samples medium, determined by of lipid recovered automated MTBE or Bligh and Dyer The are of experiments in in a To the accuracy of automated lipid of the same sample were according to either the MTBE or the Bligh and Dyer Mass spectrometric analysis of the extracts lipid species of major lipid classes and which were the of quantification of with of the total of the corresponding lipid are in in the lipid of the most abundant lipid and or for the and were we that the MTBE recipe was well suited for automated lipid extraction of biological and the same or better recoveries as the established Bligh and Dyer Recent developments in mass spectrometry enabled comprehensive quantitative profiling of eukaryotic Although lipid extraction from cells, fluids, or tissues is a in the automated lipidomics it little and the is typically to the Folch or Bligh and Dyer recipes. The MTBE extraction procedure faster and cleaner recovery of most of the major lipid classes and was well suited for shotgun in which total extracts were infused directly into a mass with The of MTBE extraction two-phase systems from the low density of the lipid-containing organic phase that forms the upper layer during phase greatly its collection and dripping losses. compared with chloroform, MTBE is and water review and approach to Health PubMed Scopus Google Scholar, H. U. of in the by the for the 2001; PubMed Scopus Google Scholar), which the as well as the health for is and forms during and of labile lipids H. M. of MTBE and Press, New Google Scholar). Rigorous testing that in biological species from major lipid classes demonstrated that the MTBE protocol similar or better compared with the Folch or Bligh and Dyer and specific limitations of the enabled processing of cells, biological fluids, and tissues and was to using a the to shotgun profiling of complex lipidomes in a automated (9.Schwudke D. Hannich J.T. Surendranath V. Grimard V. Moehring T. Burton L. Kurzchalia T. Shevchenko A. Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra.Anal. Chem. 2007; 79: 4083-4093Crossref PubMed Scopus (151) Google Scholar, 12.Ejsing C.S. Duchoslav E. Sampaio J. Simons K. Bonner R. Thiele C. Ekroos K. Shevchenko A. Automated identification and quantification of glycerophospholipid molecular species by multiple precursor ion scanning.Anal. Chem. 2006; 78: 6202-6214Crossref PubMed Scopus (331) Google Scholar, D. J. Burton L. E. Hannich J.T. C.S. Kurzchalia T. Shevchenko A. Lipid profiling by multiple precursor and neutral by the Chem. 2006; 78: PubMed Scopus Google Scholar). The are to of and and developed the for mass and to of the Kurzchalia and Shevchenko for and The Sampaio of and for of the with