Rapid nanoscale quantitative analysis of plant sphingolipid long-chain bases by GC-MS

Abstract

In eukaryotic organisms, sphingolipids are major structural lipids of biological membranes and perform additional essential functions as signalling molecules. While long-chain bases (LCB), the common precursor to all sphingolipid 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 reinvestigated and optimized a protocol destined to the quantification 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 metaperiodate, 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.

New Long Chain Bases in Lipophosphonoglycan of Acanthamoeba Castellanii

“…However, they differ from Acanthamoeba LPG in the presence of the sphingosine and sphinganine residues instead of phytosphingosine. Phytoceramide moieties structurally related to those in Acanthamoeba have been identified for GPI of amoeba Dictyostelium discoideum contact site A protein (mainly t18:0/18:1) [33], GPI S. cerevisiae proteins (mainly t18:0/24:0) [34], glycophosphosphingolipids of Leptomonas (contain t20:0 and t21:0 LCB acylated with fatty acids in the extent C [14][15][16][17][18][19][20][21][22][23][24][25][26] , but the most abundant FA are 24:0 and 18:0) [35], or in sphingolipids of Paramecium cilia (t18:0) [36]. Unlike ceramides of most protists, Acanthamoeba LPG phytoceramides are substituted with a broad spectrum of fatty acids: saturated normal and branched fatty acids (C 16 -C 28 ) and predominant normal and branched a-hydroxy fatty acids (C 20 -C 28 ) [37].…”

“…The samples of (1) native LPG and (2) LPG hydrolyzed according to the method of Watanabe et al [14] were subjected to sodium metaperiodate oxidation [15]. Briefly, native LCB was dissolved in 500 ll of methanol (5 mg per ml) and 100 ll of a freshly made 0.2 M aqueous solution of sodium metaperiodate was added [16]. The reaction mixture stood in the dark at room temperature for 1 h.…”

“…Upon separation by GC (and detection by MS), individual constituents were determined by comparison of their retention times and mass spectra with those for standards ( Fig. 4a, b [16]. The molecular ions for some constituents could be observed but they were still very weak.…”

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids

“…In animals, rafts have been implicated in a huge range of cellular processes, such as hormone signaling, membrane trafficking in polarized epithelial cells, T cell activation, cell migration, and the life cycle of influenza and human immunodeficiency viruses (Simons and Ikonen, 1997;Simons and Gerl, 2010). In plants, evidence is increasing that rafts are also involved in signal transduction processes and membrane trafficking (for review, see Mongrand et al, 2010;Simon-Plas et al, 2011;Cacas et al, 2012a).…”

Isolation and Analysis of Phospholipids in Dairy Foods

“…Therefore, to become GC compatible, these compounds have usually been hydrolyzed by phospholipase C and derivatized with tert-butyl-dimethylsilyl (TBDMS), trimethylsilyl (TMS), and pentafluorobenzoyl (PFB) [87]. On the other hand, for the analysis of sphingolipids by GC, some authors have proposed methods involving the release of the target compound using barium hydroxide, their oxidation into aldehydes by metaperiodate salts, and finally quantification of these aldehydes by GC-MS [88]. However, all these derivatization reactions add extra steps to the preparation of the samples that together with those of isolation/fractionation result in costly and cumbersome procedures.…”