Shotgun lipidomics of phosphoethanolamine-containing lipids in biological samples after one-step in situ derivatization

This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions.Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes. This article presents a novel methodology for the analysis of ethanolamine glycerophospholipid (PE) and lysoPE molecular species directly from lipid extracts of biological samples. Through brief treatment of lipid extracts with fluorenylmethoxylcarbonyl (Fmoc) chloride, PE and lysoPE species were selectively derivatized to their corresponding carbamates. The reaction solution was infused directly into the ion source of an electrospray ionization mass spectrometer after appropriate dilution. The facile loss of the Fmoc moiety dramatically enhanced the analytic sensitivity and allowed the identification and quantitation of low-abundance molecular species. A detection limitation of attomoles (amoles) per microliter for PE and lysoPE analysis was readily achieved using this technique (at least a 100-fold improvement from our previous method) with a >15,000-fold dynamic range. Through intrasource separation and multidimensional mass spectrometry array analysis of derivatized species, marked improvements in signal-to-noise ratio, molecular species identification, and quantitation can be realized. The procedure is both simple and effective and can be extended to analyze many other lipid classes or other cellular metabolites by adjustments in specific derivatization conditions. Thus, through judicious derivatization, a new dimension exploiting specific functional reactivities in each lipid class can be used in conjunction with shotgun lipidomics to penetrate farther into the low-abundance regime of cellular lipidomes. Recently, a powerful technique for the direct analysis of global cellular lipidomes [i.e., shotgun lipidomics using intrasource separation and multidimensional electrospray ionization-mass spectrometry (ESI-MS)] has emerged (see 1Han X. Gross R.W. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics.J. Lipid Res. 2003; 44: 1071-1079Google Scholar, 2Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Google Scholar, 3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar for recent reviews). In shotgun lipidomics, lipid classes in a crude lipid extract are first separated at the ion source (i.e., intrasource separation) through judicious selection of ion-pairing reagents based on the electrical properties of the lipid classes described previously (2Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Google Scholar, 4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar). The basic principles of ESI intrasource separation of lipids extend the charge separation principle of ESI, which is analogous to an electrophoretic cell, as described previously (5Tang L. Kebarle P. Effect of the conductivity of the electrosprayed solution on the electrospray current. Factors determining analyte sensitivity in electrospray mass spectrometry.Anal. Chem. 1991; 63: 2709-2715Google Scholar, 6Ikonomou M.G. Blades A.T. Kebarle P. Electrospray-ion spray: a comparison of mechanisms and performance.Anal. Chem. 1991; 63: 1989-1998Google Scholar). The involvement of ion pairing of intrasource separation is also analogous to ion-exchange chromatography, which facilitates lipid class separations (7Gross R.W. Sobel B.E. Isocratic high-performance liquid chromatography separation of phosphoglycerides and lysophosphoglycerides.J. Chromatogr. 1980; 197: 79-85Google Scholar). For electrically neutral lipids, selectivity during separations is accomplished through differential ion ratios of lipid classes in the source, depending upon the ion-pairing conditions used.Cellular lipidomes contain thousands to tens of thousands of individual molecular species of lipids. Most of these biological lipid species are linear combinations of aliphatic chains, backbones, and/or head groups, each of which represents a building block of the molecular species under consideration. Therefore, the identification of individual molecular species of the cellular lipidome can be achieved through the determination of the associated building blocks of cellular lipids. Shotgun lipidomics fulfills this task through multidimensional ESI-MS array analysis under various instrumental conditions, such as changes in ionization conditions (e.g., source temperature and spray voltage) and in fragmentation conditions [e.g., collision gas pressure, collision energy, collision gas, mass loss in neutral loss (NL) mode, and monitored ions in precursor ion (PI) mode] (3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar). Besides the basic two-dimensional (2D) mass spectral unit, in which the second dimension is constructed with specific lipid building blocks, each series of ramped changes in instrumental condition facilitates the generation of an additional dimension, each of which can potentially be used in multidimensional mass spectrometric analyses (3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar).Quantitation in shotgun lipidomics is performed by a two-step process (3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar, 8Han X. Cheng H. Mancuso D.J. Gross R.W. Caloric restriction results in phospholipid depletion, membrane remodeling and triacylglycerol accumulation in murine myocardium.Biochemistry. 2004; 43: 15584-15594Google Scholar, 9Han X. Cheng H. Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics.J. Lipid Res. 2005; 46: 163-175Google Scholar). First, the abundant molecular species in a class of polar lipids are quantitated by direct comparison of the ion peak intensities with that of a preselected amount of internal standard for the lipid class in the first-dimensional mass spectrum after correction for the 13C isotopomer differences, as described previously (2Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Google Scholar, 10Han X. Gross R.W. Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2001; 295: 88-100Google Scholar, 11Han X. Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry.Anal. Biochem. 2002; 302: 199-212Google Scholar). The foundation of this measurement is based on the observation that the ion abundance of a lipid molecular species is linearly correlated with its concentration in the sprayed solution in the low-concentration region. This observation has been validated by many independent studies (3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar, 9Han X. Cheng H. Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics.J. Lipid Res. 2005; 46: 163-175Google Scholar, 12Han X. Gross R.W. Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.Proc. Natl. Acad. Sci. USA. 1994; 91: 10635-10639Google Scholar, 13Han X. Gubitosi-Klug R.A. Collins B.J. Gross R.W. Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis.Biochemistry. 1996; 35: 5822-5832Google Scholar, 14Lehmann W.D. Koester M. Erben G. Keppler D. Characterization and quantification of rat bile phosphatidylcholine by electrospray-tandem mass spectrometry.Anal. Biochem. 1997; 246: 102-110Google Scholar, 15Kim H.Y. Wang T.C. Ma Y.C. Liquid chromatography/mass spectrometry of phospholipids using electrospray ionization.Anal. Chem. 1994; 66: 3977-3982Google Scholar, 16DeLong C.J. Baker P.R.S. Samuel M. Cui Z. Thomas M.J. Molecular species composition of rat liver phospholipids by ESI-MS/MS: the effect of chromatography.J. Lipid Res. 2001; 42: 1959-1968Google Scholar, 17Koivusalo M. Haimi P. Heikinheimo L. Kostiainen R. Somerharju P. Quantitative determination of phospholipid compositions by ESI-MS: effects of acyl chain length, unsaturation, and lipid concentration on instrument response.J. Lipid Res. 2001; 42: 663-672Google Scholar, 18Han X. Gross R.W. Specific lipid alterations in Alzheimer's disease and diabetes: shotgun global cellular lipidome analyses by electrospray ionization mass spectrometry using intrasource separation.in: Feng L. Prestwich G.D. Functional Lipidomics. Marcel Dekker, Inc., New York2005: 285-306Google Scholar). Next, the determined concentrations of these abundant molecular species are used as endogenous standards, in addition to the preselected internal standard, for ratiometric comparisons to quantitate or refine the mass content of low-abundance individual molecular species from at least one of the representative tandem mass scans for the lipid class of interest. By using tandem mass scans, the baseline fluctuations caused by chemical noise can be reduced dramatically. Thus, the measure of the linear dynamic range of ion peak intensity ratios between the selected internal standard and the unknowns of interest is expanded to penetrate into the low-abundance region of lipid molecular species directly from organic extracts. Because of the effects of differential fragmentation kinetics on different molecular species in a class (2Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Google Scholar), multiple standards, representative of the different physical properties (subclass, acyl chain length, and degree of unsaturation), have to be selected for quantitation based on tandem MS (MS/MS), as demonstrated previously (19Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar, 20Welti R. Li W. Li M. Sang Y. Biesiada H. Zhou H-E. Rajashekar C.B. Williams T.D. Wang X. Profiling membrane lipids in plant stress responses. Role of phospholipase Dα in freezing-induced lipid changes in Arabidopsis.J. Biol. Chem. 2002; 277: 31994-32002Google Scholar, 21Ekroos K. Chernushevich I.V. Simons K. Shevchenko A. Quantitative profiling of phospholipids by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer.Anal. Chem. 2002; 74: 941-949Google Scholar, 22Blom T.S. Koivusalo M. Kuismanen E. Kostiainen R. Somerharju P. Ikonen E. Mass spectrometric analysis reveals an increase in plasma membrane polyunsaturated phospholipid species upon cellular cholesterol loading.Biochemistry. 2001; 40: 14635-14644Google Scholar, 23Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Google Scholar). Thus, the determined set of individual constituents from step one represent the endogenous standards that are selected for their well-distributed changes in acyl chain length and unsaturation to appropriately cover the lipid class of interest in most cases. Of course, additional standards can be added if necessary, but in most cases, the naturally occurring distribution of acyl chain length and unsaturation is usually sufficient.In shotgun lipidomics, molecular species containing a head group of phosphoethanolamine, such as ethanolamine glycerophospholipid (PE) and lysoPE, are analyzed after the addition of a small amount of LiOH in negative ion mode (2Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Google Scholar, 4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar). These molecular species carry a weakly zwitterionic head group and thus become anionic under alkaline conditions. Unfortunately, under these experimental conditions, many low-abundance PE molecular species and almost the entire lysoPE class are buried in the baseline noise. Moreover, those PE species having high molecular weight can potentially overlap with molecular species from other lipid class(es) (e.g., phosphatidylinositol) and are difficult to identify. Furthermore, at present, there is no highly sensitive and representative MS/MS method that can be used to profile very low-abundance PE and lysoPE molecular species under the experimental conditions developed. Therefore, quantitation of very low-abundance PE molecular species and all lysoPE species is often difficult by shotgun lipidomics using the aforementioned two-step procedure.In this report, we describe a technique to identify and quantify PE and lysoPE molecular species from lipid extracts of biological tissues after a one-step derivatization in situ with 9-fluorenylmethoxylcarbonyl chloride (Fmoc-Cl) or other reagents. After derivatization, Fmoc-PE and Fmoc-lysoPE molecular species are rendered anionic and can be analyzed directly in the negative ion mode with enhanced sensitivity. Moreover, derivatization with Fmoc shifts PE molecular species out of the region where they potentially overlap with other lipid classes. Finally, product ion analysis of Fmoc-PE and Fmoc-lysoPE molecular species demonstrates a very abundant fragment corresponding to the facile NL of Fmoc. Thus, mass spectrometric analysis by NL of the Fmoc moiety from Fmoc-PE and Fmoc-lysoPE species can be used to identify and quantify lysoPE and very low-abundance PE molecular species. Collectively, this technique provides a new tool for shotgun lipidomics to penetrate farther into the low-abundance regime of PEs directly from organic extracts.MATERIALS AND METHODSMaterialsSynthetic phospholipids, including phosphatidylcholine (1,2-dimyristoleoyl-sn-glycero-3-phosphocholine; 14:1-14:1 PtdCho), phosphatidylethanolamine (1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine; 20:4-20:4 PtdEtn), phosphatidylglycerol (1,2-dipentadecanoyl-sn-glycero-3-phosphoglycerol; 15:0-15:0 PtdGro), and 1-dimyristoyl-sn-glycero-3-phosphoethanolamine (14:0 lysoPE) (used as internal standards for the corresponding lipid classes), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Fmoc-Cl and dimethylaminopyridine (DMAP) were from Sigma-Aldrich Chemical Co. (St. Louis, MO). 2-(2-Naphthyl)acetyl chloride (NAC) was purchased from Ryan Scientific, Inc. (Isle of Palms, SC). All solvents used for sample preparation and for mass spectrometric analysis were obtained from Burdick and Jackson (Honeywell International, Inc., Burdick and Jackson, Muskegon, MI). Anhydrous chloroform was purchased from Fisher Scientific, Inc. (Philadelphia, PA).Sample preparationMale mice (C57BL/6, 4 months of age) were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were killed by inhalation of carbon dioxide before tissue collection. Mouse eyes were enucleated and hemisected at the ora serrata. The cornea, iris, lens, and vitreous were removed, and the retinas were detached from the eyecups mechanically. The retina lipids were extracted by the Bligh and Dyer procedure (24Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar) with modifications as described previously (2Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of the cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Google Scholar, 4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar, 8Han X. Cheng H. Mancuso D.J. Gross R.W. Caloric restriction results in phospholipid depletion, membrane remodeling and triacylglycerol accumulation in murine myocardium.Biochemistry. 2004; 43: 15584-15594Google Scholar). Briefly, to a small volume of mouse retina homogenate containing ∼100 μg of protein (from one pair of eyes obtained from a single mouse), internal standards including 20:4-20:4 PtdEtn (48.0 nmol/mg protein) and 14:0 lysoPtdEtn (1.0 nmol/mg protein) were added just before extraction. Lipids from each mouse retina sample were extracted against 1.8 ml of 50 mM LiCl twice, back extracted against 1.8 ml of 10 mM LiCl twice, filtered with a 0.2 μm polytetrafluoroethylene syringe filter, and finally resuspended and stored in 1:1 (v/v) chloroform-methanol at an approximate concentration of 2 ml/mg protein.Next, each of the lipid extracts was individually derivatized with Fmoc-Cl as follows. Each lipid extract in 100 μl of 1:1 (v/v) chloroform-methanol containing ∼10 nmol of PE was transferred to a test tube, and the solvents were evaporated under nitrogen gas. Freshly prepared Fmoc-Cl solution in anhydrous chloroform (100 μl) was added to the test tube to make a 1:1 molar ratio of PE to Fmoc-Cl, flushed with nitrogen gas, capped, and mixed with lipids by vortexing. When other molar ratios of PE to Fmoc-Cl were used as indicated, Fmoc-Cl solutions with corresponding concentrations were also prepared before the addition of 100 μl of Fmoc-Cl solution to the reaction vessels. The test tube was kept in the dark (wrapped with foil) at room temperature for the indicated times with occasional vortexing. The reaction solution was diluted 20-fold with 1:1 (v/v) chloroform-methanol before direct infusion into an ESI mass spectrometer for lipid analysis.Samples that were derivatized with other reagents such as NAC were also prepared similarly. Briefly, freshly prepared NAC solution in anhydrous chloroform (100 μl) was added to the test tube to make a 1:1 molar ratio of PE to NAC and mixed with lipids by vortexing. When other molar ratios of PE to NAC were used, NAC solutions with corresponding concentrations were prepared. The reaction solution was diluted 20-fold with 1:1 (v/v) chloroform-methanol before direct infusion into an ESI mass spectrometer for lipid analysis.ESI-MS of lipidsESI-MS analyses were performed using a triple-quadrupole mass spectrometer (TSQ Quantum Ultra; ThermoFinnigan, San Jose, CA) equipped with an electrospray ion source, as described previously (4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar, 8Han X. Cheng H. Mancuso D.J. Gross R.W. Caloric restriction results in phospholipid depletion, membrane remodeling and triacylglycerol accumulation in murine myocardium.Biochemistry. 2004; 43: 15584-15594Google Scholar). Typically, a 1 min period of signal averaging in the profile mode was used for each MS spectrum and up to a 2 min period of signal averaging was used for each MS/MS scan. For MS/MS analysis of Fmoc-PE, the collision gas pressure was set at 1.0 mTorr and a collision energy of 30 eV or indicated values was used for NL scanning of 222.2 u; alternatively, an energy of 25 eV was used for PI scanning of fatty acyl carboxylates. Other MS/MS analysis conditions were used as described previously (4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar). As usual, plasmenylethanolamine (PlsEtn) molecular species are distinguished from plasmanylethanolamine species by comparing the mass spectra acquired from the samples before and after acid treatment (25Ford D.A. Rosenbloom K.B. Gross R.W. The primary determinant of rabbit myocardial ethanolamine phosphotransferase substrate selectivity is the covalent nature of the sn-1 aliphatic group of diradyl glycerol acceptors.J. Biol. Chem. 1992; 267: 11222-11228Google Scholar, 26Kayganich K.A. Murphy R.C. Fast atom bombardment tandem mass spectrometric identification of diacyl, alkylacyl, and alk-1-enylacyl molecular species of glycerophosphoethanolamine in human polymorphonuclear leukocytes.Anal. Chem. 1992; 64: 2965-2971Google Scholar). Alternatively, direct identification of PlsEtn molecular species in the positive ion mode is also possible (27Hsu F-F. Turk J. Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: the fragmentation processes.J. Am. Soc. Mass Spectrom. 2003; 14: 352-363Google Scholar, 28Berry K.A.Z. Murphy R.C. Electrospray ionization tandem mass spectrometry of glycerophosphoethanolamine plasmalogen phospholipids.J. Am. Soc. Mass Spectrom. 2004; 15: 1499-1508Google Scholar). It was found in this study (see Results) that PlsEtn species could be redundantly profiled by PI scanning of lysoPlsEtn ions using a collision energy of 30 eV. Identification of each ion peak corresponding to anionic lipids or Fmoc-PE species was performed in a 2D mass spectrometric array format as described previously (3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar, 4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar). Quantitation of each identified PE molecular species was conducted in a two-step procedure as described previously (3Han X. Gross R.W. Shotgun lipidomics: multi-dimensional mass spectrometric analysis of cellular lipidomes.Expert Rev. Proteomics. 2005; 2: 253-264Google Scholar, 8Han X. Cheng H. Mancuso D.J. Gross R.W. Caloric restriction results in phospholipid depletion, membrane remodeling and triacylglycerol accumulation in murine myocardium.Biochemistry. 2004; 43: 15584-15594Google Scholar, 9Han X. Cheng H. Characterization and direct quantitation of cerebroside molecular species from lipid extracts by shotgun lipidomics.J. Lipid Res. 2005; 46: 163-175Google Scholar).MiscellaneousQuantitative data from biological samples were normalized to the protein content of the tissues, and all data are presented as means ± SD of samples from 10 animals. Protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL) using BSA as a standard.RESULTSAnalysis of lipids in conventional shotgun lipidomicsBecause it is well known that the lipid profile of retinas is quite complicated (29Bell M.V. Tocher D.R. Molecular species composition of the major phospholipids in brain and retina from rainbow trout (Salmo gairdneri). Occurrence of high levels of di-(n-3)polyunsaturated fatty acid species.Biochem. J. 1989; 264: 909-915Google Scholar, 30Sellner P.A. Clough J.A. Fatty acid composition of phospholipids from chick neural retina during development.Exp. Eye Res. 1992; 54: 725-730Google Scholar), we used lipid extracts of mouse retinas as a model system to demonstrate the power of in situ derivatization for the analysis of PE and lysoPE molecular species directly from a lipid extract. ESI-MS analysis of a diluted mouse retina lipid extract directly in the negative ion mode displayed many intense ion peaks between m/z 400 and 1100 (Fig. 1A). 2D MS analysis demonstrated that most of these ions belong to anionic lipids (e.g., 15:0-15:0 PtdGro at m/z 693.5, an internal standard for anionic lipid analysis, and 18:0-20:4 phosphatidylinositol at m/z 885.6), chlorinated choline glycerophospholipid species (chlorinated 14:1-14:1 PtdCho internal standard at m/z 708.6), and chlorinated cerebrosides (e.g., chlorinated perdeuterated N-octadecanoyl cerebroside at m/z 797.7, an internal standard) in the lipid extract (Fig. 2). The 2D mass spectrometric analysis also demonstrated some PE molecular species (e.g., 20:4-20:4 PtdEtn at m/z 786.6 and 18:0-22:6 PtdEtn at m/z 790.6) in modest abundance. Abundant molecular ions corresponding to PE molecular species in the identical diluted mouse retina lipid extract were also demonstrated in the negative ion ESI mass spectrum when the sample was analyzed after the addition of a small amount of LiOH before infusion of the diluted lipid extract (Fig. 1B).Fig. 2Representative 2D ESI mass spectra of a chloroform extract of mouse retinas in negative ion mode. A conventional ESI mass spectrum was acquired in the negative ion mode directly from a diluted mouse retina lipid extract (see Fig. 1A) before analysis of lipid building blocks in the second dimension by precursor ion (PI) scanning and neutral loss (NL) scanning as indicated. Each mass spectral scan was acquired as described previously (4Han X. Yang J. Cheng H. Ye H. Gross R.W. Towards fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry.Anal. Biochem. 2004; 330: 317-331Google Scholar). All mass spectral traces were displayed after normalization to the base peak in each spectrum. IS, internal standard; m:n, acyl chain containing m carbons and n double bonds.View Large Image Figure ViewerDownload (PPT)ESI-MS analysis of Fmoc-derivatized lipidsWhen the identical lipid extract was incubated with Fmoc-Cl at a 1:1 molar ratio of Fmoc-Cl and PE for as short as 5 min and then analyzed by ESI-MS under similar conditions, the negative ion ESI mass spectrum of lipids demonstrated a totally different ion profile (Fig. 3A). Many very abundant clusters of ion peaks corresponding to the Fmoc-PE molecular species appeared in the higher mass range (m/z > 900) (Fig. 3A), whereas the abundant ion peaks corresponding to anionic lipids in Fig. 1 appeared in low or modest abundance (Fig. 3A), such as the peaks at m/z 693.5 (corresponding to 15:0-15:0 PtdGro). Many additional ion peaks were also shown in the region between m/z 700 and 850, corresponding to the chlorinated choline glycerophospholipid or cerebroside molecular species, because of the increase in chloride concentration during Fmoc-Cl treatment.Fig. 3Representative negative ion ESI-MS and product ion ESI mass spectra of a lipid extract of mouse retinas after derivatization with fluorenylmethoxylcarbonyl chloride (Fmoc-Cl). An appropriate amount of Fmoc-Cl in anhydrous chloroform was added to the identical mouse retina lipid extract used in Fig. 1 in a ratio of 1:1 [Fmoc-Cl to ethanolamine glycerophospholipid (PE) content in the extract]. The mixture was incubated at room temperature for 5 min and diluted directly with 1:1 chloroform-methanol to a concentration of ∼50 pmol/μl total lipids. The negative ion ESI mass spectrum (A) was acqui