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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, Anneke Lubben, Simon D. J. Calaminus^* and Michael J. O. Wakelam^1,*

^* Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TH, UK
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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