Elsevier

Analytical Biochemistry

Volume 378, Issue 2, 15 July 2008, Pages 197-201
Analytical Biochemistry

Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography

https://doi.org/10.1016/j.ab.2008.03.052Get rights and content

Abstract

Recent advances in research on the physiological roles of phosphoinositides in eukaryotic organisms indicate a need to distinguish molecular phosphoinositide species on the basis of their characteristic head groups as well as their glycerolipid moieties. Accurate identification of phosphoinositide species in biological samples poses an analytical challenge, because structurally similar inositol phosphate head groups must be resolved, as must lipid-associated fatty acids. Although intact phosphoinositide species have been successfully analyzed, such analyses employ state-of-the-art liquid chromatography/mass spectrometry and require expensive equipment not accessible to many researchers. Described here is a cost-efficient and reliable alternative developed by adaptation of a combination of classic methods for lipid analysis, thin-layer chromatography and gas chromatography.

Section snippets

Lipid extraction

Plant tissue (1–10 g fresh wt) was ground in liquid N2 to a fine powder using mortar and pestle. Cultured tobacco BY2 cells (Nicotiana tabacum bright yellow 2) or yeast (up to 2 g) was harvested by centrifugation. Phosphoinositides were extracted from ground powder or cell pellets by an acidic lipid extraction method allowing quantitative extraction of the anionic lipids [22]. Note that protonation of phosphoinositides was required for successful extraction and that no quantitative extraction was

Results and discussion

Because lipids are isolated from TLC plates after development, for quantitative purposes, it is critical to assess the rates of lipid recovery for this step. Equal amounts (10 μg) of various phosphoinositides were spotted onto silica plates (Merck) that were subsequently developed in different solvents. After the plates were developed, lipids were isolated and dried as described above. Identical amounts (10 μg) of control samples were added to fresh glass tubes and directly dried under streaming N

Acknowledgments

We thank Dr. Ivo Feussner (Göttingen University, Germany) for helpful discussion and critical reading of this article. We gratefully acknowledge financial support from the Union zur Förderung von Öl- und Proteinpflanzen e.V. (to M.H.) and an Emmy Noether grant from the German Research Foundation (DFG) (to I.H.).

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