Abstract
Diacylglycerol (DAG) is an important intermediate of lipid metabolism and a component of phospholipase C signal transduction. Quantification of DAG in plant membranes represents a challenging task because of its low abundance. DAG can be measured by direct infusion mass spectrometry (MS) on a quadrupole time-of-flight mass spectrometer after purification from the crude plant lipid extract via solid-phase extraction on silica columns. Different internal standards are employed to compensate for the dependence of the MS and MS/MS signals on the chain length and the presence of double bonds in the acyl moieties. Thus, using a combination of single MS and MS/MS experiments, quantitative results for the different molecular species of DAGs from Arabidopsis can be obtained.
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References
Li X, Evans J (2005) Examining the collision-induced decomposition spectra of ammoniated triglycerides as a function of fatty acid chain length and degree of unsaturation. I. The OXO/YOY series. Rapid Commun Mass Spectrom 19:2528–2538
Han X, Gross R (2001) Quantitative analysis and molecular species fingerprinting of triacylglyceride molecular species directly from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal Biochem 295:88–100
Holčapek M, Jandera P, Zderadička P, Hrubá L (2003) Characterization of triacylglycerol and diacylglycerol composition of plant oils using high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. J Chromatogr A 1010:195–215
Hutchins P, Barkley R, Murphy R (2008) Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry. J Lipid Res 49:804–813
Callender H, Forrester J, Ivanova P, Preininger A, Milne S, Brown H (2007) Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm. Anal Biochem 79:263–272
Hsu F-F, Ma Z, Wohltmann M, Bohrer A, Nowatzke W, Ramanadham S, Turk J (2000) Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation. J Biol Chem 275:16579–16589
Li YL, Su X, Stahl PD, Gross ML (2007) Quantification of diacylglycerol molecular species in biological samples by electrospray ionization mass spectrometry after one-step derivatization. Anal Chem 79:1569–1574
Bates P, Durrett T, Ohlrogge J, Pollard M (2009) Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant Physiol 150:55–72
Peters C, Li M, Narasimhan R, Roth M, Welti R, Wang X (2010) Nonspecific phospholipase C NPC4 promotes responses to abscisic acid and tolerance to hyperosmotic stress in Arabidopsis. Plant Cell 22:2642–2659
Welti R, Li W, Li M, Sang Y, Biesiada H, Zhou H-E, Rajashekar C, Williams T, Wang X (2002) Profiling membrane lipids in plant stress responses. Role of phospholipase Dα in freezing-induced lipid changes in Arabidopsis. J Biol Chem 277:31994–32002
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Browse J, Warwick N, Somerville CR, Slack CR (1986) Fluxes through the prokaryotic and eukaryotic pathways of lipid synthesis in the ‘16:3’ plant Arabidopsis thaliana. Biochem J 235:25–31
Hajra A (1974) On extraction of acyl and alkyl dihydroxyacetone phosphate from incubation mixtures. Lipids 9:502–505
Wewer V, Dombrink I, vom Dorp K, Dörmann P (2011) Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry. J Lipid Res 52:1–16
Brügger B, Erben G, Sandhoff R, Wieland FT, Lehmann WD (1997) Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc Natl Acad Sci USA 94:2339–2344
Acknowledgments
We would like to thank Helga Peisker (University of Bonn) for help during sample preparation, method development and data evaluation. This work was supported by an Instrument Grant (Forschungsgrossgeräte-Antrag) and by the Sonderforschungsbereich 645 of Deutsche Forschungsgemeinschaft.
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vom Dorp, K., Dombrink, I., Dörmann, P. (2013). Quantification of Diacylglycerol by Mass Spectrometry. In: Munnik, T., Heilmann, I. (eds) Plant Lipid Signaling Protocols. Methods in Molecular Biology, vol 1009. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-62703-401-2_5
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DOI: https://doi.org/10.1007/978-1-62703-401-2_5
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