title: Analysis of D3-,4-,5-phosphorylated phosphoinositides using HPLC.
abstract: Detection of polyphosphoinositides (PPIs) is difficult due to their low chemical abundancy. This problem is further complicated by the fact that PPIs are present as various, distinct isomers, which are difficult, if not impossible, to separate by conventional thin layer chromatography (TLC) systems. PPIs in plants include PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P 2, and PtdIns(4,5)P 2. Here, a protocol is described analyzing plant PPIs using (32)P-orthophosphorus pre-labeled material. After extraction, lipids are deacylated and the resulting glycerophosphoinositol polyphosphates (GroPInsPs) separated by HPLC using a strong anion-exchange column and a shallow salt gradient. Alternatively, PPIs are first separated by TLC, the lipids reisolated, deacylated, and the GroPInsPs then separated by HPLC.
authors.affiliation: Swammerdam Institute for Life Sciences, Section Plant Physiology, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Teun
authors.initials: T
authors.lastname: Munnik
mesh_terms: D000215:Acylation; D000837:Anion Exchange Resins; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D007295:Inositol Phosphates / Q000032:analysis; D018129:Phosphatidylinositol Phosphates / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D014316:Tritium
chemical_list: D000837:Anion Exchange Resins; D007295:Inositol Phosphates; D018129:Phosphatidylinositol Phosphates; D010761:Phosphorus Radioisotopes; C055525:phosphatidylinositol 3-phosphate; C037178:phosphatidylinositol 4-phosphate; C113567:phosphatidylinositol 5-phosphate; D014316:Tritium; C014575:glycerylphosphoinositol