HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: D600034-JLR200v1 48/1/235    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Esch, S. W. Articles by Welti, R. Search for Related Content PubMed PubMed Citation Articles by Esch, S. W. Articles by Welti, R. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 48, 235-241, January 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

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

Steven Wynn Esch^*, Pamela Tamura^*, Alexis A. Sparks^*, Mary R. Roth^*, Shivakumar P. Devaiah, Ernst Heinz, Xuemin Wang, Todd D. Williams^** and Ruth Welti^1,*

^* Kansas Lipidomics Research Center, Division of Biology, Kansas State University, Manhattan, KS 66506
Department of Biology, University of Missouri-St. Louis and Danforth Plant Science Center, St. Louis, MO 63121
Biozentrum Klein Flottbeck, University of Hamburg, D-22609 Hamburg, Germany
^** University of Kansas Mass Spectrometry Laboratory, University of Kansas, Lawrence, KS 66045

Published, JLR Papers in Press, October 19, 2006.

¹ To whom correspondence should be addressed. e-mail: welti{at}ksu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
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.

Supplementary key words phospholipid • galactolipid • oxylipins • lipidomics

Abbreviations: CID-TOF MS, collision-induced dissociation time-of-flight mass spectrometry; di15:0 PE, 1-15:0,2-15:0 phosphatidylethanolamine; ESI, electrospray ionization; FID, flame ionization detection; MGDG, monogalactosyldiacylglycerol; OPPC, 1-oleoyl (18:1),2-palmitoyl (16:0) phosphatidylcholine; POPC, 1-palmitoyl (16:0),2-oleoyl (18:1) phosphatidylcholine; POPE, 1-palmitoyl (16:0),2-oleoyl (18:1) phosphatidylethanolamine; Q-TOF, quadrupole time-of-flight

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
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 (1). Separation and analytical techniques include adsorption chromatography, silver nitrate chromatography, TLC, and HPLC (2). 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 (3–7).

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 (8). 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 (8). Here, we describe this methodology and investigate the accuracy and precision of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Lipids
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%20Polar%20Lipids">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 (9).

Plant extracts
Leaves from approximately 7 week old Arabidopsis (Columbia ecotype) plants were used for analysis. Growth conditions have been described elsewhere (8). Wounding was performed as described previously (8, 10). Arabidopsis lipids were extracted as described previously (11). 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.

CID-TOF MS
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.

CID-TOF MS data analysis
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 (12). 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. It was not necessary to correct for A + 2 isotopic overlap, because in no case did the m/z of the A + 2 peak of an examined fatty acid fall within the m/z window of the A peak of another fatty acid isotope cluster.

Gas chromatography with flame ionization detection
The solvent was completely evaporated from samples to which an internal standard of 15:0 had been added. One milliliter of 3 M methanolic hydrochloric acid was added, and the samples were bubbled with nitrogen gas and heated at 78°C for 30 min. Two milliliters of water and 2 ml of pentane were added. Samples were shaken, and the upper pentane layer was removed. This extraction was repeated twice, the solvent was evaporated, and the sample was dissolved in 150 µl of hexane.

Samples were analyzed by gas chromatography with flame ionization detection (FID) on an Agilent 6890 gas chromatograph (Agilent Technologies, Wilmington, DE) equipped with a split/splitless injector operated in the splitless mode at 250°C. The injection volume was 1 µl. The carrier gas was helium (0.7 ml/min). Compounds were separated on a Supelco 2380 [stabilized; poly(90%:10% biscyanopropyl-cyanopropylphenyl siloxane)] column (30 m x 0.25 mm inner diameter; Sigma-Aldrich, St. Louis, MO). The column temperature was ramped from 50°C to 130°C at 20°C/min, then to 250°C at 3°C/min. The FID detector was at 260°C.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
CID-TOF MS detects acyl anions from complex lipids
Fatty acyl compositional analysis by CID-TOF MS is performed by ionizing a solution of complex lipids by ESI Q-TOF MS in negative ion mode with ammonium acetate as carrier electrolyte, producing [M–H]^– and/or [M+OAc]^– acyl anions. The quadrupole is operated in radio frequency-only mode (no direct current voltage applied), allowing all lipid analytes supplied by the ion source entry into the collision cell simultaneously, regardless of size (i.e., m/z value). The acyl anions are detected by a TOF analyzer. Figure 1 shows the result of CID-TOF MS analysis performed at three different collision energies (10, 35, and 60 eV) on an extract of Arabidopsis leaves after silicic acid chromatography to reduce the free fatty acid levels in the extract. These spectra were obtained by direct infusion of the lipid extract and, as shown, are each a combination of 11 scans of 5 s duration of m/z 20–2,000.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effect of collision energy on acyl yield in collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) of plant lipids. Centroided CID-TOF analysis of column-purified polar lipids from wild-type Arabidopsis at collision energies of 10, 35, and 60 eV (spectra A, B, and C, respectively) show that total and relative anion yields change as a complex function of collision energy. All measured intensities are plotted relative to the base peak intensity for the 18:3 acyl anion at 35 eV (m/z 277). The most abundant acyl anions are labeled in C. A mass resolution slightly >6,000 is more than sufficient to achieve baseline separation of peaks for the glycerogalactose fragment of the monogalactosyldiacylglycerol (MGDG) head group (m/z 253.0923) and the nominally isobaric 16:1 acyl anion (m/z 253.2168), as shown in the inset for spectrum B (centroided data). Tiny acyl anion peaks in the 10 eV data may have been attributable to autolysis of the sample after column purification, because no expected lipid head group fragments appeared above the limits of detection to indicate that fragmentation had occurred in the collision cell at 10 eV. The acyl anion for 18:3 was chosen as the lock mass for all spectra, and each spectrum is the sum of 11 scans of 5 s duration.

CID-TOF MS fatty acyl analysis is not rigorously quantitative
Although the spectra in Fig. 1B, C are similar, the relative heights of acyl anion peaks differ in response to fragmentation at different collision energies. In Table 1 , these acyl anions are quantified and compared with the fatty acyl composition determined by fatty acid methyl ester analysis of the same Arabidopsis lipid mixture by GC with FID. By GC-FID, the detector response varies by <10% among the analyzed fatty acid methyl esters (15, 16), so GC-FID data indicate a composition close to the true composition. The spectral data produce a composition similar to, but not exactly the same as, the fatty acyl composition determined by GC-FID. Furthermore, the composition determined by CID-TOF depends somewhat on the offset voltage applied to the collision cell.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effect of polar head group and position on the glycerol backbone on acyl yield via CID-TOF. Each spectrum represents 11 scans of 5 s duration, and all spectra were acquired at 35 eV except for spectrum E, which was acquired at 10 eV. Each panel depicts a centroided CID-TOF spectrum of an equimolar mixture of 15:0, 15:0 phosphatidylethanolamine (di15:0 PE), 17:0 free fatty acid, and an additional synthetic lipid. The additional lipid in spectrum A is POPC; that in spectrum B is 1-oleoyl (18:1),2-palmitoyl (16:0) phosphatidylcholine (OPPC); that in spectrum C is 1-palmitoyl (16:0),2-oleoyl (18:1) phosphatidylethanolamine (POPE); that in spectra D and E is 1-18:1,2-16:1 MGDG. The 17:0 acyl anion was used as the common lock mass, and correction for isotopic effects was approximated by summing the monoisotopic, A + 1, and A + 2 isotope peak intensities. Resulting data for spectra A through D are summarized in Table 2, together with corresponding data taken at 60 eV and processed in a similar manner.

The data in Fig. 2 also indicate that the acyl chain position affects the CID-TOF MS spectral signal. For example, for POPC (Fig. 2A), the ratio of the peak produced by the sn2 acyl species compared with the sn1 acyl species (18:1 to 16:0) was 2.0, demonstrating a higher yield from the 2-position (Table 2). Similarly, OPPC and POPE also yielded more acyl anion from the 2-position (Fig. 2B, C, Table 2). In each of these three phospholipid cases, the more abundant acyl yield was obtained from the sn2 position, confirming observations of product ion analysis described by Murphy (17). The sn2:sn1 ratios were not identical, however, suggesting that acyl chain structure could play a secondary role in the CID-TOF yield obtained. On the other hand, the acyl yields from the 1- and 2-positions of the MGDG lipid were reversed, and the fatty acyl group in the 1-position gave a higher yield (Fig. 2D, Table 2). Guella, Frassanito, and Mancini (18) also found that fragmentation of MGDG to produce acyl anions generally occurred more readily from the sn1 than the sn2 position. These results allow us to resolve the ambiguity in a previous study (19) in which the acyl positions in naturally occurring 18:3-16:3 MGDG and 18:3-16:3 digalactosyldiacylglycerol were reported provisionally as 16:3-18:3, based on the higher yield of 18:3 acyl anion and the (incorrect) assignment of this higher-yield anion to the species in the 2-position.

The 17:0 free fatty acid, included in all of the samples in Fig. 2, was the only acyl anion recovered in substantial yield at 10 eV (Fig. 2E). This confirms the observation that MS1 spectra or spectra obtained at very low collision energies (those minimally required to usher intact ions through the collision cell) are useful for examining background levels of free fatty acids in tissue extracts (20). It should be noted that the yield of the 17:0 fatty acid was somewhat lower at 35 eV (Fig. 2A–D) than at 10 eV (all panels are on the same scale). This may reflect fragmentation by CID of the 17:0 fatty acyl anion at 35 eV, at which voltage the ratio of 17:0 to di15:0 PE is <1:1, the ratio that would be expected for the free fatty acid relative to the same molar amount of PE (Table 2).

Together, the data in Fig. 2 and Table 2 indicate that the nonquantitative nature of acyl compositional analysis by CID-TOF on biological mixtures (Table 1) stems from the different propensities of acyl chains of different lipids, and in different positions on the same lipid, to produce acyl anions. This is true at any collision offset voltage. Therefore, it is not possible to "correct" CID-TOF results by assignment of response factors to fatty acyl species, because, as shown here, response varies depending on the class and positional distribution of the fatty acid in the mixture.

Acyl species containing heteroatoms can be detected and resolved
An Arabidopsis leaf extract contains a mixture of the major plant phospholipids, PC, PE, phosphatidylglycerol, and phosphatidylinositol, the major plant galactolipids, MGDG, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, and lesser amounts of other complex lipids (21, 22). The fatty acyl composition of Arabidopsis leaves differs depending on the physiological state of the plant. For example, mechanical wounding of plant leaves has been demonstrated to result in the production of a variety of oxygenated fatty acyl species, collectively known as "oxylipins" (5, 6, 8). Oxylipins can occur both as free fatty acids or esterified to the complex polar lipids of the plant plastid (6, 8, 23–27). CID-TOF MS of an unfractionated extract of Arabidopsis leaves subjected to wounding shows that oxylipin species can be detected (Fig. 3 , Table 3 ), along with the normal chain acyl species. Although these same acyl species should be discernible, quantifiable, and resolvable by GC-MS, especially if a TOF analyzer is used, the advantages of CID-TOF MS for this analysis are its rapidity and the lack of requirement for derivatization or heating, which in some cases may lead to decomposition.

View larger version (15K): [in this window] [in a new window]   Fig. 3. CID-TOF analysis of wounded Arabidopsis. Centroided CID-TOF spectrum of Arabidopsis (Columbia ecotype) lipids at 35 eV, summary of 11 scans at 5 s/scan, after extraction from mechanically wounded leaves. Spectral data show well-defined peaks for dinor-oxophytodienoic (16:4-O; m/z 263) and oxophytodienoic (18:4-O; m/z 291) species in the extract (see Table 3 for details). The 18:3 acyl anion (theoretical m/z 277.2168) was used as lock mass.

View larger version (14K): [in this window] [in a new window]   Fig. 4. CID-TOF analysis of Arabidopsis fatty acid desaturase (fad) mutants. Centroided 35 eV CID-TOF summary spectra of Arabidopsis extracts from the wild type and three fad mutants, each spectrum is the sum of 11 scans at 5 s/scan. Spectrum A: Wild-type strain showing 16:3, 16:0, and 18:3 as the predominant acyl groups. Spectrum B: fad2 mutation, in which 18:1-to-18:2 conversion is defective, leading to the accumulation of 18:1. Spectrum C: fad3, in which 18:2-to-18:3 conversion is defective, leading to the accumulation of 18:2. Spectrum D: fad7, in which both 18:2-to-18:3 and 16:2-to-16:3 conversions are defective, causing the accumulation of 18:2 and 16:2 acyl chains. The 18:3 acyl anion was chosen as a convenient high-abundance lock mass (m/z 277.2168).

	NOTE ADDED IN PROOF

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Strictly speaking, the TOF analyzer is not a scanning device. We have used the term "scans" in connection with TOF spectra loosely in place of the more cumbersome term "duty cycle."

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the technical assistance of Bob Drake at the University of Kansas. The authors also gratefully acknowledge Ashis Nandi and Kartikeya Krothapalli (working as members of the Jyoti Shah laboratory at Kansas State University) for donation of the lipid extracts used for evaluation of the Arabidopsis mutants. The authors thank Levi Kinderknecht and Tara Thacker for assistance in growing plants. Work and instrument acquisition were supported by grants from the National Science Foundation (Grants MCB 0455318 and DBI 0521587). Support of the Kansas Lipidomics Research Center was from the National Science Foundation's Experimental Program to Stimulate Competitive Research, under Grant EPS-0236913, with matching support from the State of Kansas through the Kansas Technology Enterprise Corporation and Kansas State University, as well from National Institutes of Health Grant P20 RR-016475 from the IdeA Network of Biomedical Research Excellence program of the National Center for Research Resources. The Micromass Q-TOF-2 at the University of Kansas Mass Spectrometry Laboratory was purchased with funds from the University of Kansas, the University of Kansas Mass Spectrometry Laboratory, and the National Science Foundation Experimental Program to Stimulate Competitive Research. This is contribution 07-24-J from the Kansas Agricultural Experiment Station.

Manuscript received August 31, 2006 and in revised form October 10, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION NOTE ADDED IN PROOF REFERENCES

1. Kesselmeier, J., and E. Heinz. 1987. Separation of molecular species of plant glycolipids and phospholipids by high-performance liquid chromatography. Methods Enzymol. 148: 650–661.

2. Christie, W. W. 1982. Lipid Analysis. 2^nd edition. Pergamon Press, Oxford, England. 73–79.

3. Mueller, M. J., L. Mène-Saffrané, C. Grun, K. Karg, and E. E. Farmer. 2006. Techniques for molecular analysis. Oxylipin analysis methods. Plant J. 45: 472–489.[CrossRef][Medline]

4. Schulze, B., R. Lauchli, M. M. Sonwa, A. Schmidt, and W. Boland. 2006. Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine. Anal. Biochem. 348: 269–283.[CrossRef][Medline]

5. Vollenweider, S., H. Weber, S. Stolz, A. Chételat, and E. E. Farmer. 2000. Fatty acid ketodienes and fatty acid ketotrienes: Michael addition acceptors that accumulate in wounded and diseased Arabidopsis leaves. Plant J. 24: 467–476.[CrossRef][Medline]

6. Weber, H., B. A. Vick, and E. E. Farmer. 1997. Dinor-oxo-phytodienoic acid: a new hexadecanoid signal in the jasmonate family. Proc. Natl. Acad. Sci. USA. 94: 10473–10478.[Abstract/Free Full Text]

7. Hamberg, M., A. Sanz, and C. Castresana. 1999. -Oxidation of fatty acids in higher plants. Identification of a pathogen-inducible oxygenase (piox) as an -dioxygenase and biosynthesis of 2-hydroperoxylinolenic acid. J. Biol. Chem. 274: 24503–24513.[Abstract/Free Full Text]

8. Buseman, C. M., P. Tamura, A. A. Sparks, E. J. Baughman, S. Maatta, J. Zhao, M. R. Roth, S. W. Esch, J. Shah, T. D. Williams, et al. 2006. Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves. Plant Physiol. 142: 28–39.[Abstract/Free Full Text]

9. Heinz, E. 1971. Semisynthetic galactolipids of plant origin. Biochim. Biophys. Acta. 231: 537–544.[Medline]

10. Laudert, D., and E. W. Weiler. 1998. Allene oxide synthase: a major control point in Arabidopsis thaliana octadecanoid signalling. Plant J. 15: 675–684.[CrossRef][Medline]

11. Welti, R., W. Li, M. Li, Y. Sang, H. Biesiada, H. E. Zhou, C. B. Rajashekar, T. D. Williams, and X. Wang. 2002. Profiling membrane lipids in plant stress responses. Role of phospholipase D in freezing-induced lipid changes in Arabidopsis. J. Biol. Chem. 277: 31994–32002.[Abstract/Free Full Text]

12. Savitzky, A., and M. J. E. Golay. 1964. Smoothing and differentiation of data by simplified least squares procedures. Anal. Chem. 36: 1627–1639.[CrossRef]

13. Morris, H. R., T. Paxton, A. Dell, J. Langhorne, M. Berg, R. S. Bordoli, J. Hoyes, and R. H. Bateman. 1996. High sensitivity collisionally-activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 10: 889–896.[CrossRef][Medline]

14. Bristow, A. W. 2006. Accurate mass measurement for the determination of elemental formula—a tutorial. Mass Spectrom. Rev. 25: 99–111.[CrossRef][Medline]

15. Christie, W. W. 1991. Gas chromatographic analysis of fatty acid methyl ester with high precision. Lipid Technology. 3: 97–98.

16. Dodds, E. D., M. R. McCoy, L. D. Rea, and J. M. Kennish. 2005. Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry. Lipids. 40: 419–428.[CrossRef][Medline]

17. Murphy, R. C. 1993. Mass spectrometry of lipids. In Handbook of Lipid Research. Vol. 7. Plenum Press, New York. 223–226.

18. Guella, G., R. Frassanito, and I. Mancini. 2003. A new solution for an old problem: the regiochemical distribution of the acyl chains in galactolipids can be established by electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17: 1982–1994.[CrossRef][Medline]

19. Welti, R., W. Li, M. Li, Y. Sang, H. Biesiada, H. Zhou, C. B. Rajashekar, T. D. Williams, and X. Wang. 2002. Profiling membrane lipids in plant stress responses. Role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J. Biol. Chem. 277: 31994–32002.[Abstract/Free Full Text]

20. Han, X., and R. W. Gross. 2003. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J. Lipid Res. 44: 1071–1079.[Abstract/Free Full Text]

21. Zien, C. A., C. Wang, X. Wang, and R. Welti. 2001. In vivo substrates and the contribution of the common phospholipase D, PLD, to wound-induced metabolism of lipids in Arabidopsis. Biochim. Biophys. Acta. 1530: 236–248.[Medline]

22. Devaiah, S. P., M. R. Roth, E. Baughman, M. Li, P. Tamura, R. Jeannotte, R. Welti, and X. Wang. 2006. Quantitative profiling of polar glycerolipid species from organs of wild-type Arabidopsis and a PHOSPHOLIPASE D1 knockout mutant. Phytochemistry. 67: 1907–1924.[CrossRef][Medline]

23. Stelmach, B. A., A. Müller, P. Hennig, S. Gebhardt, M. Schubert-Zsilavecz, and E. W. Weiler. 2001. A novel class of oxylipins, sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogalactosyl diglyceride, from Arabidopsis thaliana. J. Biol. Chem. 276: 12832–12838. [Erratum. 2001. J. Biol. Chem. 276: 28628.][Abstract/Free Full Text]

24. Hisamatsu, Y., N. Goto, K. Hasegawa, and H. Shigemori. 2003. Arabidopsides A and B, two new oxylipins from Arabidopsis thaliana. Tetrahedron Lett. 44: 5553–5556.[CrossRef]

25. Hisamatsu, Y., N. Goto, M. Sekiguchi, K. Hasegawa, and H. Shigemori. 2005. Oxylipins arabidopsides C and D from Arabidopsis thaliana. J. Nat. Prod. 68: 600–603.[CrossRef][Medline]

26. Creelman, R. A., and J. E. Mullet. 1997. Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 355–381.[CrossRef][Medline]

27. Shah, J. 2005. Lipids, lipases, and lipid-modifying enzymes in plant disease resistance. Annu. Rev. Phytopathol. 43: 229–260.[CrossRef][Medline]

28. Kunst, L., J. Browse, and C. Somerville. 1988. Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proc. Natl. Acad. Sci. USA. 85: 4143–4147.[Abstract/Free Full Text]

29. Kunst, L., J. Browse, and C. Somerville. 1989. Altered chloroplast structure and function in a mutant of Arabidopsis deficient in plastid glycerol-3-phosphate acyltransferase activity. Plant Physiol. 90: 846–853.[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				H. Maeda, T. L. Sage, G. Isaac, R. Welti, and D. DellaPenna Tocopherols Modulate Extraplastidic Polyunsaturated Fatty Acid Metabolism in Arabidopsis at Low Temperature PLANT CELL, February 1, 2008; 20(2): 452 - 470. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: D600034-JLR200v1 48/1/235    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Esch, S. W. Articles by Welti, R. Search for Related Content PubMed PubMed Citation Articles by Esch, S. W. Articles by Welti, R. Social Bookmarking                      What's this?