Analysis of intact phosphoinositides in biological samples

It is now apparent that each of the known, naturally occurring polyphosphoinositides, the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates [PtdIns(3,4) P-2, PtdIns(3,5) P-2, PtdIns(4,5) P-2], and phosphatidylinositol trisphosphate [PtdIns(3,4,5) P-3], have distinct roles in regulating many cellular events, including intracellular signaling, migration, and vesicular trafficking. Traditional identification techniques require [P-32] inorganic phosphate or [H-3] inositol radiolabeling, acidified lipid extraction, deacylation, and ion-exchange head group separation, which are time-consuming and not suitable for samples in which radiolabeling is impractical, thus greatly restricting the study of these lipids in many physiologically relevant systems. Thus, we have developed a novel, high-efficiency, buffered citrate extraction methodology to minimize acid-induced phosphoinositide degradation, together with a high-sensitivity liquid chromatography-mass spectrometry (LC-MS) protocol using an acetonitrile-chloroform-methanol-water-ethylamine gradient with a microbore silica column that enables the identification and quantification of all phosphoinositides in a sample. The liquid chromatograph is sufficient to resolve PtdInsP(3) and PtdInsP(2) regioisomers; however, the PtdInsP regioisomers require a combination of LC and diagnostic fragmentation to MS3. Data are presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples.