Analysis of Phosphoinositides from Complex Plant Samples by Solid-Phase Adsorption Chromatography and Subsequent Quantification via Thin-Layer and Gas Chromatography.
Methods Mol Biol
; 2295: 379-389, 2021.
Article
em En
| MEDLINE
| ID: mdl-34047988
ABSTRACT
The determination of phosphoinositide molecular species in plant material is challenging because of their low abundance concurrent with a very high abundance of other membrane lipids, such as plastidial glycolipids. Phosphoinositides harbor an inositol headgroup which carries one or more phosphate groups at different positions of the inositol, linked to diacylglycerol via a phosphodiester. Thus, a further analytical challenge is to distinguish the different inositol-phosphate headgroups as well as the fatty acids of the diacylglycerol backbone. The method presented in this chapter expands on previous protocols for phosphoinositide analysis by employing chromatographic enrichment of phospholipids and their separation from other, more abundant lipid classes, before analysis. Lipids extracted from plant material are first separated by solid-phase adsorption chromatography into fractions containing neutral lipids, glycolipids, or phospholipids. Lipids from the phospholipid fraction are then separated by thin-layer chromatography (TLC) according to their characteristic head groups, and the individual phosphatidylinositol-monophosphates and phosphatidylinositol-bisphosphates are isolated. Finally, the fatty acids associated with each isolated phosphatidylinositol-monophosphate or phosphatidylinositol-bisphosphate are analyzed in a quantitative fashion using gas chromatography (GC). The analysis of phosphoinositides by this combination of methods provides a cost-efficient and reliable alternative to lipidomics approaches requiring more extensive instrumentation.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfatidilinositóis
/
Cromatografia
/
Lipídeos de Membrana
Idioma:
En
Revista:
Methods Mol Biol
Ano de publicação:
2021
Tipo de documento:
Article