Rapid characterization of the fatty acyl composition of complex lipids by collision-induced dissociation time-of-flight mass spectrometry

Abstract

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for ∼1 min per sample. Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for ∼1 min per sample. Fatty acyl composition is most commonly analyzed by gas chromatography after derivatization to fatty acid methyl esters. When flame ionization or a mass spectrometer is used as the detector, quantitative compositional data are obtained. Liquid chromatography provides an alternative means of separation, in which fatty acids can be detected by ultraviolet light absorption after chemical conversion to an appropriate derivative form (1Kesselmeier J. Heinz E. Separation of molecular species of plant glycolipids and phospholipids by high-performance liquid chromatography..Methods Enzymol. 1987; 148: 650-661Crossref Scopus (8) Google Scholar). Separation and analytical techniques include adsorption chromatography, silver nitrate chromatography, TLC, and HPLC (2Christie W.W. Lipid Analysis. 2nd. Pergamon Press, Oxford, England1982: 73-79Google Scholar). Most gas chromatography and liquid chromatography protocols for fatty acyl species are optimized for normal chain fatty acyl species. Other fatty acids, such as those containing heteroatoms (e.g., oxygen), often require specialized procedures for analysis. Plant oxylipins, for example, have been analyzed by GC-MS or liquid chromatography after various extraction and derivatization steps designed to enable detection and minimize the degradation of these often low-level compounds (3Mueller M.J. Mène-Saffrané L. Grun C. Karg K. Farmer E.E. Techniques for molecular analysis. Oxylipin analysis methods..Plant J. 2006; 45: 472-489Crossref PubMed Scopus (79) Google Scholar, 4Schulze B. Lauchli R. Sonwa M.M. Schmidt A. Boland W. Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine..Anal. Biochem. 2006; 348: 269-283Crossref PubMed Scopus (46) Google Scholar, 5Vollenweider S. Weber H. Stolz S. Chételat A. Farmer E.E. Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves..Plant J. 2000; 24: 467-476Crossref PubMed Google Scholar, 6Weber H. Vick B.A. Farmer E.E. Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family..Proc. Natl. Acad. Sci. USA. 1997; 94: 10473-10478Crossref PubMed Scopus (270) Google Scholar, 7Hamberg M. Sanz A. Castresana C. α-Oxidation of fatty acids in higher plants. Identification of a pathogen-inducible oxygenase (piox) as an α-dioxygenase and biosynthesis of 2-hydroperoxylinolenic acid..J. Biol. Chem. 1999; 274: 24503-24513Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Recently, we used a novel negative ion mode mass spectrometric scanning method for rapid profiling of free and ester-linked fatty acyl moieties in crude lipid extracts (8Buseman C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. et al.Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves..Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (162) Google Scholar). The collision-induced dissociation time-of-flight (CID-TOF) experiment involved simultaneous collision-induced dissociation of all members of a complex mixture of lipids in the collision cell of a quadrupole time-of-flight (Q-TOF) instrument. The TOF analyzer provided accurate mass characterization of all intact acyl fragments in a single spectrum. Using this method as a “discovery scan,” we identified fatty acyl groups, including several oxygenated species that increased in response to wounding of the plant Arabidopsis thaliana (8Buseman C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. et al.Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves..Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (162) Google Scholar). Here, we describe this methodology and investigate the accuracy and precision of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS). 1-15:0,2-15:0 phosphatidylethanolamine (di15:0 PE), POPC, 1-oleoyl (18:1),2-palmitoyl (16:0) phosphatidylcholine (OPPC), and 1-palmitoyl (16:0),2-oleoyl (18:1) phosphatidylethanolamine (POPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). 17:0 fatty acid was purchased from NuChek Prep, Inc. (Elysian, MN). 1-18:1,2-16:1 monogalactosyldiacylglycerol (MGDG) was prepared semisynthetically (9Heinz E. Semisynthetic galactolipids of plant origin..Biochim. Biophys. Acta. 1971; 231: 537-544Crossref PubMed Scopus (24) Google Scholar). Leaves from approximately 7 week old Arabidopsis (Columbia ecotype) plants were used for analysis. Growth conditions have been described elsewhere (8Buseman C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. et al.Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves..Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (162) Google Scholar). Wounding was performed as described previously (8Buseman C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. et al.Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves..Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (162) Google Scholar, 10Laudert D. Weiler E.W. Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling..Plant J. 1998; 15: 675-684Crossref PubMed Google Scholar). Arabidopsis lipids were extracted as described previously (11Welti 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: 31994-32002Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Seeds of the act1-1, fad2-1, fad3-2, and fad7-1(gl1) mutants of Arabidopsis were obtained from the Arabidopsis Biological Resource Center at Ohio State University (catalog Nos. CS200, CS8041, CS8034, and CD3108, respectively). Because a high-throughput analytical approach is being developed, extracts were analyzed in crude form, without further processing, except as noted. Crude extracts containing the lipid from ∼2 mg leaf biomass (dry weight)/ml chloroform were diluted 100-fold into the infusion solvent before mass analysis. For purposes of analyzing collision energy effects only, a crude extract of 7 week old Arabidopsis (Columbia ecotype) was further processed to reduce levels of naturally occurring free fatty acids. Crude extract (500 μl in chloroform) was passed through an activated silicic acid (Unisil; Clarkson Chemical Co., Williamsport, PA) column (0.3 g suspended in chloroform and packed into a glass Pasteur pipette) and washed with 8 ml of chloroform to elute the neutral lipids. Polar lipids were then eluted with 8 ml of chloroform-methanol (2:1), the solvent was evaporated to dryness, and the lipids were dissolved in 1 ml of chloroform. This, in turn, was diluted ∼100-fold into the electrospray solvent before mass analysis. Electrospray ionization (ESI)-CID-TOF MS spectra were acquired with a Micromass Q-TOF-2 tandem mass spectrometer (Micromass, Ltd., Manchester, UK). The TOF analyzer was tuned for maximum resolution (10,000 resolving power) with argon in the collision cell. The pusher frequency was set at 22 kHz for a mass range of m/z 20–1,000 and at 16 kHz for a mass range of m/z 20–2,000. Mass selection in the Q1 quadrupole was turned off, whereas the collision cell remained at the specified voltage offset. Unselected precursor ions were subjected to CID in the negative mode. Micromass MassLynx software was used as the operating software. All TOF spectra were acquired with daily mass calibration. Samples were dissolved in chloroform-methanol-300 mM ammonium acetate in water (60:133:7, v/v/v) and infused into the ESI source of the Q-TOF at 20–40 μl/min. Careful selection of background subtraction, smoothing, and centroiding parameters was required for reproducible processing of CID-TOF raw continuum spectral data. Spectrum manipulation software was used to attenuate chemical background noise to ∼10 counts per ion by setting a linear horizontal baseline subtraction threshold to remove the lowest 40% of signals in the continuum spectrum. After baseline subtraction, the spectral data were subjected to two successive smooths of ±3 “time bin” channels (0.0625 u/channel) according to the Savitzky-Golay method (12Savitzky A. Golay M. J.E. Smoothing and differentiation of data by simplified least squares procedures..Anal. Chem. 1964; 36: 1627-1639Crossref Scopus (14792) Google Scholar). Smoothed peaks were centroided at a minimum peak width at half height of four channels (0.25 units). These manipulations were adjusted by trial and error to ensure one centroid “stick” per resolvable ion in this region. Spectra were mass-corrected by locking on an appropriate known anion in the m/z region of interest. Chemical formulas for the product ions were determined using the Micromass MassLynx chemical formula tool limiting formula to C, H, N, O, P, S, sensible valence, and rings plus double bonds restrictions. For calculation of fatty acid composition, the centroided, mass-corrected spectral data were transferred to an Excel data template with a “lookup” function that selected mass/signal pairs within specified m/z windows for the expected and identified acyl anions. The intensities of acyl-specific signals were summed, divided by the total signal, and multiplied by 100 to obtain mol% of total fatty acids. was to for in the m/z of the peak of an fatty acid within the m/z of the peak of fatty acid The solvent was evaporated from samples to which an of been of acid was and the samples were with gas and at for of water and ml of were Samples were and the was extraction was the solvent was and the was dissolved in μl of Samples were analyzed by gas chromatography with flame ionization detection on an gas with a in the mode at The was 1 The gas was were on a column The column was from to at then to at The detector was at Fatty acyl compositional analysis by CID-TOF MS is performed by a of complex lipids by ESI Q-TOF MS in negative ion mode with ammonium acetate as acyl anions. The quadrupole is in mode voltage all lipid by the ion source into the collision cell of m/z The acyl anions are detected by a TOF 1 the of CID-TOF MS analysis performed at collision and on an extract of Arabidopsis after silicic acid chromatography to reduce the free fatty acid levels in the These spectra were obtained by infusion of the lipid extract as are a of of of m/z 20–2,000. When the voltage on the collision cell was levels of acyl anions be detected in this free fatty acids. when higher collision were to the collision negative anions were produced and The spectra obtained at and acyl anions as well as These data that the acyl anions from CID of the complex lipids. The accurate mass of the TOF analyzer to nominally isobaric species A. J. M. J. tandem mass spectrometry on a novel time-of-flight mass Mass PubMed Scopus Google Scholar, mass for the of 2006; PubMed Scopus Google Scholar). For example, as shown in the to the spectral peak at m/z to the chemical formula which is a fragment derived from is from the peak at m/z to the chemical formula which is a acyl with the the obtained at total ion in the peaks in the m/z range of interest. Although the spectra in are the of acyl anion peaks in response to at collision 1 these acyl anions are and with the fatty acyl composition determined by fatty acid methyl analysis of the Arabidopsis lipid mixture by with the detector response by the analyzed fatty acid methyl W.W. analysis of fatty acid methyl with Scholar, M.R. of fatty acid methyl flame ionization detection mass PubMed Scopus Google data a composition to the The spectral data a composition the the fatty acyl composition determined by the composition determined by CID-TOF on the voltage to the collision acid by CID-TOF with a double from that with a double collision-induced dissociation gas chromatography with flame ionization of Arabidopsis lipids were analyzed by CID-TOF at and were used to fatty acid methyl esters. data are means in mol% were to the total detected for the acyl in with a double from that with a double in a new collision-induced dissociation gas chromatography with flame ionization of Arabidopsis lipids were analyzed by CID-TOF at and were used to fatty acid methyl esters. data are means in mol% were to the total detected for the acyl in Polar lipids of known composition and were used to the of lipid on acyl anion peak in CID-TOF MS spectra at was by the of the and the acyl chain position on the glycerol the CID-TOF MS derived from a mixture of POPC, and 17:0 fatty The of the acyl peak derived from to the of the peak from was peak peak as shown in the obtained conditions with and acyl composition as the complex lipid in with the acyl and 17:0 fatty The of the peak derived from to the peak from was the as in be expected of complex lipids with the the of the acyl anion peaks produced from and were The of the and acyl anion peaks were these intensities and that at collision complex lipids containing and to approximately the and to a lipids containing The of and lipids to acyl anions was at at which voltage the of and to were the chain data for lipid at and Lipid Lipid 1-15:0,2-15:0 1-oleoyl (18:1),2-palmitoyl (16:0) 1-palmitoyl (16:0),2-oleoyl (18:1) anion data were to the total for all four acyl in CID-TOF of a single and 17:0 free fatty acid were of lipids POPC, and were as described in the for The acyl at the position of lipid is total lipid for lipids is as the of the and acyl in a new 1-15:0,2-15:0 1-oleoyl (18:1),2-palmitoyl (16:0) 1-palmitoyl (16:0),2-oleoyl (18:1) anion data were to the total for all four acyl in CID-TOF of a single and 17:0 free fatty acid were of lipids POPC, and were as described in the for The acyl at the position of lipid is total lipid for lipids is as the of the and acyl The of acyl anion yield from have from of precursor ion ionization in negative mode. For example, in mM (60:133:7, ESI of ions to a and and the for in the of is that at electrospray the and produced acyl anions with intensities by a of acyl anions derived from the various in a biological mixture all be expected to to the spectrum. The in signal produced by species with and species in this that single collision energy is for the quantitative of acyl from lipid The data in that the acyl chain position the CID-TOF MS spectral For example, for the of the peak produced by the acyl species with the acyl species to was a higher yield from the and acyl anion from the C, of these the acyl yield was obtained from the of product ion analysis described by Mass spectrometry of of Lipid Press, Scholar). The were that acyl chain a in the CID-TOF yield obtained. the the acyl from the and of the lipid were and the fatty acyl in the a higher yield and R. new for an old the of the acyl in galactolipids can be by electrospray ionization tandem mass Mass PubMed Scopus Google that of to acyl anions from the the These to the in a R. Li W. Li M. Sang Y. Biesiada H. Zhou H. Rajashekar C.B. Williams T.D. Wang X. Profiling membrane lipids in plant stress responses. Role of phospholipase in freezing-induced lipid changes in Arabidopsis..J. Biol. Chem. 2002; 277: 31994-32002Abstract Full Text Full Text PDF PubMed Scopus Google in which the acyl in naturally occurring and were as on the higher yield of acyl anion and the of this anion to the species in the The 17:0 free fatty in all of the samples in was the acyl anion in yield at the that spectra or spectra obtained at collision required to intact ions through the collision are for background levels of free fatty acids in extracts X. of from crude extracts of biological samples by ESI mass a to Lipid Full Text Full Text PDF PubMed Scopus (687) Google Scholar). be that the yield of the 17:0 fatty acid was at at are on the by CID of the 17:0 fatty acyl anion at at which voltage the of 17:0 to is the that be expected for the free fatty acid to the of the data in and that the of acyl compositional analysis by CID-TOF on biological from the of acyl of and in on the to acyl anions. is at collision is to CID-TOF by of response to fatty acyl as shown response on the class and of the fatty acid in the Arabidopsis leaf extract a mixture of the major plant and the major plant and and of complex lipids Wang C. Wang X. R. and the of the phospholipase to of lipids in Biophys. Acta. PubMed Scopus Google Scholar, Roth M.R. Baughman E. Li M. Tamura P. R. R. Wang X. profiling of species from of Arabidopsis and a 2006; PubMed Scopus Google Scholar). The fatty acyl composition of Arabidopsis on the of the For example, wounding of plant been to in the of a of oxygenated fatty acyl known as S. Weber H. Stolz S. Chételat A. Farmer E.E. Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves..Plant J. 2000; 24: 467-476Crossref PubMed Google Scholar, 6Weber H. Vick B.A. Farmer E.E. Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family..Proc. Natl. Acad. Sci. USA. 1997; 94: 10473-10478Crossref PubMed Scopus (270) Google Scholar, C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. et al.Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves..Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (162) Google Scholar). can as free fatty acids or to the complex lipids of the plant H. Vick B.A. Farmer E.E. Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family..Proc. Natl. Acad. Sci. USA. 1997; 94: 10473-10478Crossref PubMed Scopus (270) Google Scholar, C.M. Tamura P. Sparks A.A. Baughman E.J. Maatta S. Zhao J. Roth M.R. Esch S.W. Shah J. Williams T.D. et al.Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves..Plant Physiol. 2006; 142: 28-39Crossref PubMed Scopus (162) Google Scholar, B.A. A. P. S. M. Weiler E.W. novel class of oxylipins, from Arabidopsis Biol. Chem. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. K. H. and two new oxylipins from Arabidopsis Scopus Google Scholar, Y. M. K. H. and from Arabidopsis PubMed Scopus Google Scholar, J.E. and of in Plant Physiol. Plant Biol. 1997; PubMed Scopus Google Scholar, J. Lipids, and in plant PubMed Scopus Google Scholar). CID-TOF MS of an extract of Arabidopsis subjected to wounding that species can be detected with the normal chain acyl species. Although these acyl species be and resolvable by a TOF analyzer is the of CID-TOF MS for this analysis are and the of for derivatization or which in to analysis of wounded Chemical in of lipid extract of wounded Arabidopsis was subjected to CID-TOF and increased levels of oxylipins with wounding were detected within an m/z range of of the were the for of all acyl detected by this in a new lipid extract of wounded Arabidopsis was subjected to CID-TOF and increased levels of oxylipins with wounding were detected within an m/z range of of the were the for of all acyl detected by this the precision of the samples were prepared for CID-TOF MS from the lipid The extract was derived from the of which a in an that in of lipids L. J. C. of lipid biosynthesis in a of Arabidopsis in Natl. Acad. Sci. USA. PubMed Google Scholar, L. J. C. and function in a of Arabidopsis in Physiol. PubMed Scopus Google Scholar). the levels of and energy was set at of the higher total signal obtained at this voltage with CID-TOF MS were from and the were The data are in The species shown for of the acyl species for species in the precision of the was precision of CID-TOF precision was by CID-TOF of the lipid The extract was derived from the of which a in the of lipids and levels of and of were used for the analysis of The acyl of the of all acyl anions for species in were to within a of an acyl anion in trial required that the m/z within of the m/z the acyl anion to in 1 of the For the in which the anion was the for the was in a new precision was by CID-TOF of the lipid The extract was derived from the of which a in the of lipids and levels of and of were used for the analysis of The acyl of the of all acyl anions for species in were to within a of an acyl anion in trial required that the m/z within of the m/z the acyl anion to in 1 of the For the in which the anion was the for the was CID-TOF MS can be used to biological samples for in fatty acid For example, that fatty acid desaturation in Arabidopsis are by CID-TOF mass spectra of extracts from Arabidopsis and fatty acid mutants the higher levels of the acyl chain the as a of to to an increased of and increased levels of and with levels in The of CID-TOF MS to and normal chain fatty acyl species and acyl species containing heteroatoms this for rapid screening of biological samples to and conditions that in acyl species alterations. is that samples are identified by CID-TOF MS screening as being from control or a analysis of these samples be by MS CID-TOF MS methodology provides a rapid screening tool the detection and accurate of acyl species from biological and analysis steps are Although the data detection of samples with in fatty acid composition, which enable the of or alterations. the TOF analyzer is a scanning have used the in with TOF spectra in of the The the of at the University of The and as members of the Shah at State for of the lipid extracts used for of the Arabidopsis The and for in plants. and were by from the and of the Center was from the to with from the State of through the and State as well from of from the of of the Center for The Micromass Q-TOF-2 at the University of Mass was purchased with from the University of the University of Mass and the to is from the collision-induced dissociation time-of-flight mass spectrometry 1-15:0,2-15:0 phosphatidylethanolamine electrospray ionization flame ionization detection monogalactosyldiacylglycerol 1-oleoyl (18:1),2-palmitoyl (16:0) phosphatidylcholine 1-palmitoyl (16:0),2-oleoyl (18:1) phosphatidylcholine 1-palmitoyl (16:0),2-oleoyl (18:1) phosphatidylethanolamine quadrupole time-of-flight