The analysis of intact phosphoinositides in biological samples

Trevor R. Pettitt, Stephen K. Dove, Anneke Lubben, Simon D. J. Calaminus, and Michael J. O. Wakelam

Institute for Cancer Studies, Birmingham University, Birmingham B15 2TT

Corresponding Author: m.j.o.wakelam{at}bham.ac.uk

It is now apparent that each of the known, naturally occurring polyphosphoinositides - the phosphatidylinositol monophosphates (PtdIns3P, PtdIns4P, PtdIns5P), phosphatidylinositol bisphosphates (PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2) and phosphatidylinositol trisphosphate (PtdIns(3,4,5)P3), have distinct roles in regulating many cellular events, including intracellular signaling, migration and vesicular trafficking. Traditional identification techniques require [32P]Pi or [3H]inositol radiolabelling, acidified lipid extraction, deacylation and ion-exchange headgroup separation which are time consuming and not suitable for samples where radiolabelling 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 minimise acid-induced phosphoinositide degradation, together with a high sensitivity, liquid chromatography-mass spectrometry (LC-MS) protocol utilising 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 LC is sufficient to resolve PtdInsP3 and the PtdInsP2 regio-isomers, however the PtdInsP regio-isomers require a combination of LC and diagnostic fragmentation to MS3. Data is presented using this approach for the analysis of phosphoinositides in human platelet and yeast samples

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