ORIGINAL PAPER Rapid nanoscale quantitative analysis of plant sphingolipid long-chain bases by GC-MS Jean-Luc Cacas & Su Melser & Frédéric Domergue & Jérôme Joubès & Brice Bourdenx & Jean-Marie Schmitter & Sébastien Mongrand Received: 24 January 2012 / Revised: 14 April 2012 / Accepted: 18 April 2012 / Published online: 11 May 2012 # Springer-Verlag 2012 Abstract In eukaryotic organisms, sphingolipids are major structural lipids of biological membranes and perform addi- tional essential functions as signalling molecules. While long-chain bases (LCB), the common precursor to all sphin- golipid classes, is represented by only one major molecular species in animals and fungi, up to nine LCB have been found in plants. In the absence of genuine plant sphingolipid references required for proper quantification, we have rein- vestigated and optimized a protocol destined to the quanti- fication of total plant LCB that relies on the use of gas chromatography-mass spectrometry (GC-MS). This rapid three-step protocol sequentially involves (1) the release of LCB from biological samples using barium hydroxide solution, (2) their oxidation into aldehydes by metaperio- date, and (3) the subsequent identification/quantification of these aldehydes by GC-MS. It is simple and reliable and enables separation of aldehydes upon their stero- specificity. It further enables the quantification of total LCB from a wide variety of samples including yeast and animal cell cultures. Keywords GC . Bioanalytical methods . Biological samples Electronic supplementary material The online version of this article (doi:10.1007/s00216-012-6060-1) contains supplementary material, which is available to authorized users. J.-L. Cacas (*) : S. Melser : F. Domergue : J. Joubès : B. Bourdenx : S. Mongrand Laboratoire de Biogenèse Membranaire, UMR 5200 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France e-mail: [email protected]J.-M. Schmitter Chimie Biologie des Membranes et Nanoobjets (CBMN)-UMR 5248, Centre de Génomique Fonctionnelle, Université de Bordeaux, BP 68, Université Bordeaux 2-Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France Present Address: J.-L. Cacas Centre INRA de Dijon, UMR 1347 Agroécologie, ERL6300 CNRS/INRA/Université de Bourgogne, Pole Interaction Plante-Microorganisme (IPM), 17 Rue Sully, 21000 Dijon, France Present Address: S. Melser Laboratoire MRGM, Rare Diseases: Genetics and Metabolism, Université Bordeaux 1–2, Deuxième Etage, Ecole des Sages-Femmes, CHU Bordeaux, France Present Address: B. Bourdenx Fermentalg SA, 4 bis rue Rivière, 33500 Libourne, France Anal Bioanal Chem (2012) 403:2745–2755 DOI 10.1007/s00216-012-6060-1
Transcript
ORIGINAL PAPER
Rapid nanoscale quantitative analysis of plant sphingolipidlong-chain bases by GC-MS
Jean-Luc Cacas & Su Melser & Frédéric Domergue &
Jérôme Joubès & Brice Bourdenx &
Jean-Marie Schmitter & Sébastien Mongrand
Received: 24 January 2012 /Revised: 14 April 2012 /Accepted: 18 April 2012 /Published online: 11 May 2012# Springer-Verlag 2012
Abstract In eukaryotic organisms, sphingolipids are majorstructural lipids of biological membranes and perform addi-tional essential functions as signalling molecules. Whilelong-chain bases (LCB), the common precursor to all sphin-golipid classes, is represented by only one major molecularspecies in animals and fungi, up to nine LCB have beenfound in plants. In the absence of genuine plant sphingolipidreferences required for proper quantification, we have rein-vestigated and optimized a protocol destined to the quanti-fication of total plant LCB that relies on the use of gaschromatography-mass spectrometry (GC-MS). This rapidthree-step protocol sequentially involves (1) the release ofLCB from biological samples using barium hydroxide
solution, (2) their oxidation into aldehydes by metaperio-date, and (3) the subsequent identification/quantificationof these aldehydes by GC-MS. It is simple and reliableand enables separation of aldehydes upon their stero-specificity. It further enables the quantification of totalLCB from a wide variety of samples including yeast andanimal cell cultures.
Electronic supplementary material The online version of this article(doi:10.1007/s00216-012-6060-1) contains supplementary material,which is available to authorized users.
J.-L. Cacas (*) : S. Melser : F. Domergue : J. Joubès :B. Bourdenx : S. MongrandLaboratoire de Biogenèse Membranaire,UMR 5200 CNRS-Université Victor Segalen Bordeaux 2,146 rue Léo Saignat,33076 Bordeaux, Francee-mail: [email protected]
J.-M. SchmitterChimie Biologie des Membranes et Nanoobjets (CBMN)-UMR5248, Centre de Génomique Fonctionnelle,Université de Bordeaux,BP 68, Université Bordeaux 2-Victor Segalen,146 rue Léo Saignat,33076 Bordeaux Cedex, France
Present Address:J.-L. CacasCentre INRA de Dijon, UMR 1347 Agroécologie,ERL6300 CNRS/INRA/Université de Bourgogne,Pole Interaction Plante-Microorganisme (IPM),17 Rue Sully, 21000 Dijon, France
Present Address:S. MelserLaboratoire MRGM, Rare Diseases: Genetics and Metabolism,Université Bordeaux 1–2,Deuxième Etage, Ecole des Sages-Femmes,CHU Bordeaux, France
Present Address:B. BourdenxFermentalg SA, 4 bis rue Rivière,33500 Libourne, France
Sphingolipids are widely distributed within the living world.This family of lipids plays a conserved dual role acrosskingdoms as structural lipids and signalling molecules.Sphingolipids are essential components of the plasma mem-brane and nanoscale subcompartments of the plasma mem-brane, known as membrane rafts [1, 2]. Some of these lipidsare also found in the endomembrane system where they areinvolved in maintaining homeostasis through regulating cellsecretion activity [3–5]. Beside their structural role, sphin-golipids were further shown to participate in the control ofautophagy and cell fate under stress conditions in animaland yeast cells [6, 7]. They were also proposed to do so inplant systems [8, 9].
Distinct classes can be distinguished among sphingoli-pids. Long-chain bases (LCB), an 18-carbon-long aliphaticchain bearing an amine group and two or three hydroxylgroups, form one such class and represent the conservedprecursors of all other sphingolipid classes. PhosphorylatedLCB are well-described and conserved signalling moleculesacross kingdoms [10]. In animals, sphingosine (d18:14(E)) isthe main LCB whereas phytosphingosine (t18:0) predomi-nates in yeast. In plants, the LCB content is much morecomplex, being composed of up to eight LCB derived fromthe precursor D-erythro-sphinganine (d18:0). Sphinganinecan be either hydroxylated or desaturated on the C-4 posi-tion and/or desaturated on the C-8 position, explaining thewider complexity of plant LCB (Fig. 1). Overall, saturatedor mono-unsaturated tri-hydroxylated compounds (t18:0and t18:1, respectively) account for the main plant LCB(for review, refer to [11]). The ceramide class of sphingolipids,known for nearly two decades for its role in animal cellapoptosis [12], is composed of a LCB moiety to which a(very-) long-chain fatty acid has been amidified. Finally,glycosylated sphingolipids, which probably represent themost diversified class of lipids on earth, result from the ester-ification of one or multiple sugar moieties either directly to theceramide molecules or through an intermediate inositolphosphate group linked to the ceramide via an ester bond.The latter class encompasses subcategories such as themonohexosyl-ceramides, animal-specific polysaccharidic-ceramides (cerebrosides, globosides, gangliosides, andsulfatides), and inositol phosphate-containing sphingolipids(known as glycosyl-inositolphosphoryl-ceramides, or GIPC),which are only found in fungal and plant organisms (forreview, refer to [13]). Despite a growing body of studies, theexact implication of these complex sphingolipids in cellularprocesses remains elusive in plants [13].
During the last decade, the sphingolipid field has beenvery busy. From a technical perspective, such a keen interesthas led to the development of powerful sphingolipidomicstrategies relying on the use of liquid chromatography-
electrospray ionization-tandem mass spectrometry, andsphingolipid contents have been approached at the scale ofmolecular species in many organisms, including animals,fungi, and plants [14–16]. Alternative strategies for estimat-ing sphingolipid steady-state levels do exist and rely onindirect quantification via the total LCB concentrations.Although reducing sphingolipidome complexity, these GC-and liquid chromatography (LC)-based methods were alreadyproven instrumental in the plant biology field [17–19]. Indeed,since each plant shingolipid class exhibits a specific LCBcomposition, the overall sphingolipid profile can be deducedfrom the LCB content. With the current genetic dissection ofthe plant sphingolipid biosynthesis and signalling pathways,such analytical methods are becoming all the more critical.However, they need to be fast and simple for screeningmutants and determining molecular phenotype. This situationelicited us to reinvestigate and optimize a GC-based methodpreviously described [20, 21].
Nearly 50 years ago, Sweeley and Moscatelli [20]reported on a protocol destined to estimate the LCB levelsof lipid extracts isolated from blood plasma, brain, and yeastsamples. This protocol relied on the oxidation of free LCBinto long-chain fatty aldehydes, the amount of which wasdetermined by gas–liquid partition chromatography. Morerecently, Bonavanture and coworkers [21] briefly describeda similar protocol using GC analysis for measuring the LCBconcentration of leaf tissues. We have optimized such aquantitative method because gas chromatography-massspectrometry (GC-MS) equipment is easy to handle andcommon to most lipid laboratories. This rapid three-stepprotocol sequentially involves (1) the release of LCB usingbarium hydroxide, (2) their oxidation into aldehydes bymetaperiodate, and (3) the quantification of these aldehydesby GC-MS. This protocol, modified to make it simpler andfaster, was successfully applied to the quantitative analysisof total LCB from a wide variety of samples includingplant tissues from several species, yeast, and animal cellcultures, purified lipid fractions as well as purified complexsphingolipids.
Methods
Chemicals
All solvents used in this study were LC-grade. Bariumhydroxide (B2507), primuline (P-7522), and pyridiniumchlorochromate (190144) were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). The saturatedbarium hydroxide solution (10%, w/v) was prepared usingdistilled water and stored at room temperature. Sodiummetaperiodate (NaIO4) was purchased from Merck(K22282297; Fontenay-sous-Bois, France).
2746 J.-L. Cacas et al.
Lipids used in this study
Soybean glucosylceramide (Glucer, 131304P), standarddihydrosphingosine (sphinganine, d20:0, 860674P), andphytosphingosine (t17:0, 860602P) were purchased fromAvanti Polar Lipids, Inc. (Alabaster, Alabama, USA). Stan-dard sphingosine (d14:14(E), 1833) was purchased fromBiovalley (Matreya, Inc., Pleasant Gap, Pennsylvania,USA). Phytosphingosine (t18:0, P2795), sphinganine (d18:0,D3314), sphingosine (d18:14(E), S7174), and primary fattyalcohols from tetradecan-1-ol to triacontan-1-ol (185388,258741, 258768, 234494, 169102, L3507, H2139, O3379,T3777) were purchased from Sigma-Aldrich (Saint-QuentinFallavier, France). All fatty aldehydes, except for the verylong-chain aldehydes, were also purchased from Sigma-Aldrich: nonanal (W278203), 6(Z)-nonenal (W358002), 2(E)-nonenal (W321303), 2,6(E,E)-nonadienal (W376604),and 2,6(E,Z)-nonadienal (294675) (Electronic supplementarymaterial Table S1).
Synthesis of long-chain fatty aldehydes
Primary fatty alcohols were oxidized into the correspondinglong-chain fatty aldehydes (from 14- to 30-carbon-long;
Electronic supplementary material Table S1) using pyridi-nium chlorochromate as described in [22].
Living materials and growth conditions
Tobacco plants (Nicotiana tabacum cv. Xanthi and Nicotia-na benthamiana) were grown in a growth chamber at 25 °Cunder 16/8-h day/night conditions. Arabidopsis thaliana(Col-0) plants were grown in a growth chamber under shortday conditions (10-h photoperiod) at 22/20 °C day/nightwith 80% relative humidity. Alfalfa (Medicago truncatula)plants were grown as previously described [19]. TobaccoBY2 (N. tabacum cv. Bright Yellow 2) and A. thaliana(accession Landsberg erecta) cell cultures were grown aspreviously described, respectively [23, 24]. The yeast dele-tion strain sur2Δ (YDR297w; BY4742 genetic background)was obtained from the EUROpean Saccharomyces cerevisiaeARchive for Functional analysis (EUROSCARF). Yeast cellswere grown overnight in YPDmedium at 30 °C. Upon assess-ing cell culture density by means of the optical density at600 nm, cells were harvested by centrifugation at 4 °C, andpellets were stored at −20 °C until use. Henrietta Lacks(HeLa) cells were plated at 104 cells/cm2 and kept for 48 hin glucose-containingmedium [25]. This medium consisted of
Fig. 1 Plant LCB and their fatty aldehyde derivatives resulting frommetaperiodate oxidation. Nine LCB presented in this figure have beenreported in plants. The precursor–product relationship between LCB isindicated by arrows. Dihydroxylated LCBs were previously shown to
be oxidized into shorter fatty aldehydes with (n-2) carbon atomswhereas oxidation of trihydroxylated LCB gives (n-3) carbon-longaldehydes [20]. LCB-derived aldehydes are presented in rectanglesnext to the corresponding LCB
Quantification of sphingolipid LCB by GC-MS 2747
high-glucose DMEM (Invitrogen 11960044) containing25 mM glucose, supplemented with 10 mM HEPES, 1 mMsodium pyruvate, and 10% FBS. Cultures were kept in 5%CO2 at 37 °C. HeLa cells were harvested by scrapping andcentrifuged before washing with PBS buffer. Pellets were keptat −20 °C until use.
Plasma membrane and detergent-insoluble membranepurification
Plasma membrane (PM) fractions were purified as previous-ly described [26]. Fully developed tobacco leaves from 8-week-old plants or 7-day-old BY2 cell cultures were usedfor that purpose. Detergent-insoluble membranes (DIMs)were prepared from microsomal fractions extracted fromroot tissues of 4-week-old alfalfa plants as described inBorner et al. [18].
Plant sphingolipid extraction and purification
GIPC were extracted as previously described [27]. Briefly,this method inspired from Carter and Koob [28] relies on thedifference in solubility between GIPC and glycerolipid inacidic 70% ethanol at −20 °C. Upon overnight incubation at−20 °C, centrifugation at 4 °C allows for differential sedi-mentation of GIPC and glycerolipids. The pellet is thenresuspended in tetrahydrofuran/MetOH/H2O (4:4:1, v/v/v)containing 0.1% formic acid and warmed at 60 °C for 5–10 min to allow the full solubilization of GIPC. The last stepconsists of a two-phase water/butan-1-ol (1:1, v/v) partition-ing. GIPC are recovered in the butanol phase. GIPC sampleswere stored at 4 °C until use. Before LCB analysis, theGIPC-containing butanol phase was dried under nitrogenflux. The dry residue was used for treatment with the bariumhydroxide solution.
Separation of long-chain bases by thin-layerchromatography
LCB were separated using the following solvent system,CHCl3/MetOH/NH4OH 2 N (40:10:1, v/v/v). They werevisualized under UV light upon staining with a primulinesolution (0.025 mg/mL) freshly prepared in acetone/water,8:2 (v/v). Thin-layer chromatography (TLC) was used foroptimizing the extraction and oxidation steps of the protocol(see the “Results and discussion” section). LCB releasedand extracted from plant leaf tissues and yeast cell cultureswere also chromatographed before oxidation in order to getrid of molecules that may interfere with the metaperiodate-mediated oxidation step or coelute with standards. HPTLCsilica plates (10×10 cm, Silica gel 60, Merk) werepurchased from VWR International (Fontenay-sous-Bois,France).
Release and extraction of LCB from biological samples
LCB were released from biological samples by direct over-night incubation at 110 °C in 1 mL dioxane and 1 mL 10%(w/v) Ba(OH)2 solution prepared in water. Limited amountsof starting material were sufficient for proper quantificationof LCB—15 μg of GIPC, 100 μg protein equivalent of PMand DIM, 2.5–10 mg dry weight (DW) of plant tissues, 2.5–10 mg DW of yeast cells, and 100 mg fresh weight (FW) ofHeLa cells.
Upon addition of 6 mL distilled water, LCB wereextracted from the barium hydroxide/dioxane/water mixturewith 4 mL of diethylether. This extraction step was repeatedonce. The two diethylether phases were then pooled anddried under nitrogen flux. Depending on samples, dry resi-dues were either directly treated with metaperiodate orresuspended in methanol and chromatographed on thinlayers. Remarkably, diethylether phases can be stored at4 °C for a day without any loss in LCB yield.
Oxidation of long-chain bases into fatty aldehydes
The 0.2 M working solution of metaperiodate (NaIO4) wasprepared with distilled water immediately before use. Oxida-tion was carried out as previously described [29]. Dry residuesobtained upon LCB extraction from purified lipids (GIPC),membrane fractions (PM and DIM), and HeLa cells weredirectly dissolved in 1 mL methanol. One hundred microlitersof the metaperiodate solution were then added, and the mixturewas incubated at room temperature in the dark for 1 h undermild shaking. Dry ether residues obtained from plant tissuesand yeast cells were resuspended in 100 μL of methanol andloaded onto silica plates to be resolved in the conditionsmentioned above. Plates were immersed in a primuline solu-tion. The migration behavior of LCB was deduced from that ofauthentical standards belonging to the different classes(d14:1(4E), d20:0, and t17:0; Electronic supplementary materialTable S2). Portions of the plates containing LCB werescrapped off, and the resulting silica was incubated for 1 h atroom temperature under mild shaking in 1 mL methanol and100 μL of the metaperiodate working solution. LCB-derivedlong-chain fatty aldehydes were then extracted into 1 mLhexane following addition of 1 mL water. To concentratesamples before injection, the aldehyde-containing hexanephase was dried under nitrogen flux at room temperature, andaldehydes were finally redissolved in 100 μL hexane. Onceextracted in hexane, aldehydes were stable at 4 °C over a week.
Identification and quantification of long-chain fattyaldehydes by GC-MS
For the separation of LCB-derived fatty aldehydes, a 30 m×250 μm HP-5MS capillary column (5% phenyl-methyl-
2748 J.-L. Cacas et al.
siloxane, 0.25 μm film thickness, Agilent) was used withhelium carrier gas at 2 mL/min; injection was in splitlessmode; injector and mass spectrometry (MS) detector tem-peratures were set to 250 °C (Agilent 6850 coupled to amass analyzer Agilent 6975); the oven temperature was heldat 50 °C for 1 min and then programmed with a 25 °C/minramp to 150 °C (2 min hold), a 10 °C/min ramp to 210 °C,and 75 °C/min ramp to 320 °C (5 min hold).
Upon separation by GC and detection by MS, the ioncurrent of each molecular species of interest was determinedand further used for calculating the amount of molecules bycomparison with the appropriate internal standards. Theseresults were expressed in micrograms. Taking into accountthe molecular weight of individual LCB-derived aldehydes,the quantity in moles for each molecular species was calcu-lated and expressed as mole percent. Retention indexes werecalculated using the following formula:
Ix ¼ 100n þ 100 tx � tnð Þ= tnþ1 � tnð Þ;where tx is the retention time of a given aldehyde, tn is theretention time of the normal aldehyde with n carbon atoms,and tn+1 is the retention time of the normal aldehyde with(n+1) carbon atoms, all retentions times being measuredunder conditions of linear temperature increase. The limitof detection (LOD) was defined as the lowest concentra-tion with a signal-to-noise ratio equal to 3, and the limitof quantification (LOQ) was defined as the lowest con-centration with a signal-to-noise ratio equal to 10.
Results and discussion
Identification of LCB-derived fatty aldehydes by gaschromatography-mass spectrometry
In the present work, the LCB amount is indirectly measuredvia the quantification of LCB-derived fatty aldehydesobtained by oxidation with sodium metaperiodate. Dihy-droxylated LCB were previously shown to be oxidized intoshorter fatty aldehydes with (n-2) carbon atoms whereasoxidation of trihydroxylated LCB gives (n-3) carbon-longaldehydes [20]. This reaction principle is illustrated with thenine LCB found in plant cells (Fig. 1).
As commercially available standards mostly consist ofshort-chain aldehydes, saturated long-chain fatty aldehydes(from 14 to 30 carbons) were synthesized as described in thesection “Methods.” GC retention times observed under con-ditions of linear temperature programming for normal saturatedfatty aldehydes were characterized by a linear relationshipversus carbon chain length (regression coefficient of 0.99; seeElectronic supplementary material Table S1). We thus usedthese saturated aldehydes as references for the determinationof retention indexes (Fig. 2). These retention data, combined
with characteristic ion fragments obtained by electron impactionization (see below), were used for identifying fatty alde-hydes from biological samples (Fig. 2).
Fragmentation of fatty aldehydes after electron impactionization
Using electron impact ionization, normal fatty aldehydes arereadily identified by a series of characteristic ions: losses ofwater ([M-18]+.) and ethylene ([M-28]+.), occurrence of[M-44]+., [M-46]+., and m/z 55 fragment ions. A fragmenta-tion scheme is presented on Fig. S1 (Electronic supplementarymaterial). Representative mass spectra of chosen aldehydesare also available as Electronic supplementary material(Fig. S2). When the alkyl chain length increases, the relativeabundance of [M-28]+. and [M-44]+. diminish, whereas theabundance of the [M-18]+. ion increases. Starting from the[M-46]+. ion in the direction to lower mass values, a series ofions with even m/z values and 14 Da spacing is observedfurther, and ion at m/z 44 is also found, but the abundance ofthis latter ion decreases when the length of the alkyl chainincreases (Electronic supplementary material Fig. S2a, b).
Electron impact mass spectra of aldehydes bearing anunsaturation in position 2 of the alkyl chain still showthe occurrence of [M-18]+. and 55 ions, but [M-28]+.,[M-44]+., and [M-46]+. are either weak or missing(Electronic supplementary material Fig. S2c). Comparedwith the normal aldehydes having the same length, theretention index of theses unsaturated aldehydes showsan increase of about 177 units (Fig. 2).
Mass spectra of aldehydes bearing an unsaturation inposition 6 of the alkyl chain are characterized by the occur-rence of [M-18]+., [M-44]+., and 55 ions, whereas [M-46]+.ions are missing. Furthermore, a series of homologous ionswith decreasing even m/z values starts from the [M-44]+.ion (Electronic supplementary material Fig. S2d). Comparedwith the normal aldehyde having the same length, the reten-tion index of theses aldehydes shows a decrease of about 20units (Fig. 2).
When two unsaturations occur in the alkyl chain, massspectra of aldehyde show the presence of [M-18]+., with astrong abundance of m/z 55 and 70 ions, whereas [M-28]+.,[M-44]+., and [M-46]+. are either very weak or absent(Electronic supplementary material Fig. S2e). Comparedwith the normal aldehyde having the same length, the reten-tion index of aldehydes with two unsaturations shows anincrease of about 57 units (Fig. 2).
Quantitative aspects of the GC method
Linear range within which our GC method allows forthe proper quantification of LCB-derived aldehydes wasdetermined using a set of commercially available short-
Quantification of sphingolipid LCB by GC-MS 2749
chain standard aldehydes (Electronic supplementary materialTable S1). Nonanal, 2(E)-nonenal, and 2,6(E,E)-nonadienalwere chosen as representatives of the saturated, mono-, anddi-unsaturated fatty aldehydes families, respectively. Datapresented in the Electronic supplementary material TableS3 show that our method enables the quantification of nona-nal over nearly three orders of magnitude, from about 0.8 to500 ng/μL (i.e., from 5.6 pmoles/μL to 3.5 nmoles/μL). Alinear response within the same concentration range wasalso found for the two other tested aldehydes (Electronicsupplementary material Table S3). The LOD and theLOQ determined for nonanal were close to 0.1 and0.8 ng/μL, respectively. With respect to previous works
where LCB were derivatized with O-phthaldialdehydebefore being quantified by high-performance liquid chro-matography (HPLC) with a fluorescence detector [17,30], our method appears to display a broader dynamicrange and the combination of both retention and massspectra data provides safe structure assignments of alde-hydes. If required, a lower LOQ could be reached viachemical derivatization and selected ion monitoring [31].
Qualitative aspects of the GC method
Because naturally occurring stereo-isomers of LCB mightperform distinct signalling functions and reflect the level of
Fig. 2 Representative GCtraces corresponding to HeLacells (a), tobacco leaf tissues(b), and yeast sur2Δ cells (c).The three panels show resultsobtained using the GC methoddescribed in the “Methods”section. Biological sampleswere treated as described inFig. 3. Fatty aldehydes havebeen numbered using Arabicnumbers. These numbers referto the lower panel table (d),where retention times, as wellas calculated retention indexes,are mentioned for eachLCB-derived fatty aldehyde.Standard aldehydes areindicated by stars (a–c). Whilethe main LCB in animal cells isknown to be sphingosine(a, 10), yeast sur2Δ mutantstrain exhibits strongaccumulation of 18- and20-long carbon dihydroxylatedsphinganines (c, 8 and 12,respectively). In tobaccoplants, the LCB distribution isdominated by trihydroxylatedmono-unsaturated and saturatedLCB (4 and 5, respectively),as well as dihydroxylateddiunsaturated LCB (9)
2750 J.-L. Cacas et al.
different sphingolipid classes, it is of utmost importance toseparate them in order to get a proper quantification.While shotgun (electrospray ionization-mass spectrometry(ESI-MS)) requires a time-consuming pre-separation LCstep to do so, HPLC methods [17, 30] present the advan-tage of the direct processing of samples. Similarly, our GCmethod was set to enable sharp and direct separation offatty aldehydes upon their stereo-specificity. In samplesdisplaying high LCB complexity, like those prepared fromplant leaf tissues, we indeed managed to quantify levels oftwo stereo-isomer couples: 6(Z)- and 6(E)-hexadecenal, aswell as 6(Z)- and 6(E)-pentadecenal (Fig. 2b). We alsoidentified and quantified 2(E),6(Z,E)-hexadecadienal com-pounds corresponding to d18:2 LCB. The second doublebond was not assigned any conformation as both stero-isomers display the same fragmentation scheme (Electronicsupplementary material Fig. S1), and there is no commer-cially available genuine standard. In addition, to the bestof our knowledge, no informative reports were found inthe literature that indicate the main d18:2 isomers presentin plant systems. Hence, our method allows the identifi-cation and quantification of eight out of the nine plantLCBs. It further allows the handling of many samples aday due to a full GC-MS run duration of approximately20 min.
Optimization of the protocol
Having established quantitative and qualitative aspects re-lated to the GC method, we proceeded to optimize previ-ously described protocols [20, 21]. Because acidic lysis hasproven difficult to handle due to LCB lability under suchconditions [20, 32], an alkaline treatment using bariumhydroxide was our preferred choice for LCB release fromsphingolipids. Using glucosylceramides (10 μg) as LCBsource, various conditions of LCB hydrolysis were tested.Overnight incubation in 1:1 (v/v) mixture of dioxane andaqueous Ba(OH)2 (10%, w/v) at 110 °C was found optimalfor the digest of the lipid (Electronic supplementary materialFig. S3). When applied to biological samples, these condi-tions enabled a quantitative estimation of LCB using asstarting material as little as 5 mg DW of plant leaf tissues,2.5 mg DW of yeast cells, and 80 mg FW of in vitro humancell cultures.
Upon alkaline release from sphingolipids, free LCB wereextracted from the barium hydroxide/dioxane mixture. Thisstep of the protocol was slightly modified by replacingpetroleum ether [20] or chloroform [21] with diethylether.Compared with petroleum ether, the latter solvent exhib-its a higher purity. It is also lighter than chloroform,which allowed the recovery of LCB in the upper etherphase and prevented contaminations from the dirty in-terphase when pipetting. Two successive extractions of
the aqueous phase with diethylether were sufficient forextracting all free LCB.
In previous study [21], compounds that might interferewith metaperiodate oxidation or coelute with long-chainfatty aldehydes in GC (like pigments) were removed bysuccessive washings with HCl and KOH. Although this stepresulted in full loss of interfering molecules, the LCB re-covery was poor in our hands (about 10%). To overcomethis problem, samples were chromatographed on thin-layersilica plates; portions of the lanes containing LCB were thenscrapped off and directly treated with metaperiodate asdescribed in the “Methods” section. This short extra step(no more than 30 min) was only applied to plant leaf tissueand yeast cell samples.
Finally, the question as to whether several internal stand-ards were required for the proper quantification of LCB wastackled using commercially available LCB (t17:0, d14:14(E),d20:0). These standards were simultaneously added to thebarium/dioxane mixture and further processed using ouroptimized protocol (see Fig. 3; for the full description ofthe protocol, refer to the corresponding “Methods” sec-tions). While nearly hundred percent of d20:0 was recov-ered, the recovery yield of the two other LCB was waybelow with 70% for d14:14(E) and 24% for t17:0 (Electronicsupplementary material Fig. S4). In view of theseresults, the use of all three commercial LCB as internalstandards is recommended for the assay, except whenyeast samples are analyzed as they contain minor quan-tities of the endogenous LCB d20:0. In this case, theamount of the latter LCB is calculated taking into ac-count the level of d14:14(E) standard. Based on theseexperiments and those conducted on biological samples(Tables 1 and 2), repeatability and reproductibility of ourmethod were calculated for each class of LCB. In detail,while the repeatability averages 17% for saturated dihy-droxylated LCB, that of tri-hydroxylated and unsaturateddihydroxylated LCB is about 5.5 and 8%, respectively.Overall, reproductibility was similar for all studied LCBwith an accuracy of 1.7% for saturated dihydroxylatedLCB, 2% for tri-hydroxylated LCB, and 2.4% for unsat-urated dihydroxylated LCB. Furthermore, quantities ofLCB extracted from biological samples were in goodcorrelation with previously published data. For example,while we extracted 3.69 nmol total LCB/mg DW of A.thaliana leaves, Markham and coworkers [17] reportedon 1.92 nmol/mg DW.
In order to validate our protocol and assess its robustness,a broad range of plant samples was processed and resultswere compared with published data (Table 1). Overall, theLCB composition of all tested samples was found similar, ifnot identical, to that obtained using a HPLC with fluores-cence detector, ESI-MS, or GC-flame ionization detection(FID) technologies. For instance, the large predominance of
Quantification of sphingolipid LCB by GC-MS 2751
t18:0 and t18:1 in GIPC sphingolipids purified from leavesof the genetically tractable plant model A. thaliana wasretrieved using our protocol. Results obtained for purifiedlipid fractions, including PM and DIM from distinct plantsources, only showed faint differences with those of theliterature. In the former case, such a discrepancy might beexplained by differences in purity of PM samples. In thelatter case, minor LCB (d18:0, d18:1 and d18:2) that werenot previously reported [19] were observed in our samples,illustrating the better sensitivity of our GC method com-pared with that involving the successive derivatization ofLCB with O-phthaldialdehyde, separation by HPLC, andfluorescence measurement.
Because sharp quantification of LCB from unprocessedcrude samples is crucial for many physiological approachesin biology, plant leaf tissues, yeast and mammalian cellswere also tested in this study. Although slight, but signif-icant, dissimilarities in the LCB profiles of plant leavesexist between our data and those of others [17, 21, 33],they are unlikely due to technical issues but rather reflectdifferences in the physiological status of plants betweenstudies. Indeed, the fact that several samples allow for a
good match between our results and the literature supportssuch a hypothesis. This is perfectly exemplified with theyeast deletion mutant strain sur2Δ, which is deficient forthe C4-hydroxylase of LCB and exhibits a characteristicLCB content dominated by dihydroxylated compounds(d18:0 and d20:0).
Additional biological samples of interest, whose LCBcompositions have not yet been characterized, were alsoprocessed following our protocol (Fig. 3) in order to illus-trate its feasibility (Table 2). As expected, N. benthamianatobacco leaves showed a quite similar LCB compositionwhen compared with that of N. tabacum tobacco leaves(Table 1). By contrast, the widely spread tobacco cell culturemodel, Bright Yellow 2 (BY2), exhibited a peculiar profilewhere di-unsaturated LCB were absent. Interestingly, theLCB contents of both GIPC and plasma membrane puri-fied from BY2 cells were comparable, suggesting thatGIPC are the major sphingolipids of the plasma membranein this model. Finally, immortalized human HeLa cells, theoldest cell line used in the cancer research field, displayeda LCB profile with one major species, i.e., sphingosine(d18:14(E)).
Samples
Release of LCB
Extraction of LCB
Injection
GC-MS
Oxidation of LCB
into fatty aldehydes
Extraction of
fatty aldehydes
Concentration of
fatty aldehydes
Animal, yeast and plant cells, plant tissues, purified membrane fractions (PM & DIM) or purified lipids
+ 3 vol. H2O + 2 vol. diethylether, centrifugation, back extraction with 2 vol. diethylether, centrifugation, pooled diethylether phases brought to dryness
+ 1 mL methanol + 100 L NaIO4(0.2M in H2O), incubation in the dark at room temperature for 1 hour
+ 1 mL H2O + 1 mL hexane, shaking, centrifugation
upper hexane phase dried, dry residue resuspended in 100 L hexane
TLC (optional)
Internal standards
+
(t17:0, d20:0, d14:1)
Fig. 3 Optimized protocolfor LCB analysis by GC-MS.Results presented in this studywere obtained using thisprotocol. Except for yeast andplant leaf tissues that require anextra-TLC step for removingcontaminating compounds, allsamples were treated followingthe linear graph chart. Forfurther technical details, refer tothe corresponding sections ofthe “Methods” part
2752 J.-L. Cacas et al.
Conclusions
In this study, a protocol dedicated to the quantification oftotal sphingolipid LCB by GC-MS was reinvestigatedand optimized. This protocol relies on the sequentialrelease of LCB using barium hydroxide solution, theiroxidation into aldehydes by metaperiodate, and subse-quent quantification of these aldehydes by GC-MS. Itenables the quantification of total LCB from a broad
variety of samples including plant tissues, yeast andanimal cells, purified lipid fractions, as well as extractedpurified sphingolipids. It is simple, fast, and reliable,requires limited amount of starting material, and providessuperior selectivity compared with regular protocolsbased on LC use. For these reasons, we believe that thisprotocol will be useful for forward and reverse geneticapproaches that are currently carried out in the plantsphingolipid field.
Table 2 Total LCB contents from uncharacterized biological samples
To illustrate the feasibility of our protocol, samples that were not previously characterized in the literature were prepared as described in the“Methods” section and processed following the graph chart presented in Fig. 3. GIPC were purified for 7-day-old BY2 cell cultures. LCBcomposition of A. thaliana and N. tabacum cells was also determined from 7-day-old cell cultures. LCB content of N. benthamiana leaf tissues wasassessed using fully developed leaves of 4-week-old plants; d18:1 is the sum of different stereo-isomers, i.e., d18:14(E) , d18:18(E) , and d18:18(Z) . Meanand SD of three independent experiments
Solidus symbol not detected
Table 1 Comparison of results obtained in this study by quantitative GC-MS to previously published data
Solidus symbol not detected; d18:1 is the sum of different stereo-isomers, i.e., d18:14(E) , d18:18(E) , and d18:18(Z) . Mean and SD of threeindependent experimentsa No standard deviation given in the cited referenceb Refers to a personnal communication from Brace and Rudin
Quantification of sphingolipid LCB by GC-MS 2753
Acknowledgments We would like to warmly thank Dr. ChristelleGuillier (INRA, Dijon, France) for providing alfalfa DIM samples. Weare grateful to Dr. Charles Rudin and Dr. Jenni Brace (University ofChicago, Chicago, IL, USA) for sharing results about the LCBcomposition of yeast sur2Δ mutants. We also wish to warmly thankDr. Eric Brenner (Université de Strasbourg, Strasbourg, France) andKaren Gaudin (Université Bordeaux 2, Bordeaux, France) for enthusi-astic and stimulating discussions. We thank Dr. François-Xavier Felpin(Université Bordeaux 1, Bordeaux, France) for helping with thesynthesis of long-chain fatty aldehydes. We are grateful to Dr. PatrickMoreau, Dr. René Lessire, and Dr. Jean-Jacques Besoule (CNRS,Bordeaux, France) for critical reading and stimulating discussions.We acknowledge the platforms Métabolome-Lipidome-Fluxome ofBordeaux (https://www.bordeaux.inra.fr/umr619/RMN_index.htm)for contribution to mass spectrometry equipment. This work has beenfunded by the French National Agency for Research, ANR program(NT09_517917, ANR PANACEA to SM and JMS). We declare noconflict of interest with any work cited in this study.
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