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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.D600004-JLR200 on April 21, 2006

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Journal of Lipid Research, Vol. 47, 1588-1596, July 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Methods

Analysis of intact phosphoinositides in biological samples

Trevor R. Pettitt*, Stephen K. Dove{dagger}, Anneke Lubben§, Simon D. J. Calaminus* and Michael J. O. Wakelam1,*

* Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TH, UK
{dagger} School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
§ Bruker Daltonics Limited, Coventry CV4 9GH, UK

Published, JLR Papers in Press, April 21, 2006.

1 To whom correspondence should be addressed. e-mail: 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]inorganic phosphate or [3H]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 PtdInsP3 and PtdInsP2 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.

Abbreviations: LC-MS, liquid chromatography-mass spectrometry; PtdIns, phosphatidylinositol; PtdInsP, phosphatidylinositol monophosphate; PtdInsP2, phosphatidylinositol bisphosphate; PtdInsP3, phosphatidylinositol 3,4,5-trisphosphate

Supplementary key words lipidomics • mass spectrometry • liquid chromatography-mass spectrometry • platelets • yeast


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