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Journal of Lipid Research, Vol. 46, 1796-1802, August 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

A targeted mass spectrometric analysis of phosphatidylinositol phosphate species

Stephen B. Milne^*, Pavlina T. Ivanova^*, Dianne DeCamp, Robert C. Hsueh and H. Alex Brown^1,*

^* Department of Pharmacology and Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, Nashville, TN 37232
Alliance for Cellular Signaling Cell Core Laboratory, University of Texas Southwestern Medical Center, Dallas, TX 75390

Published, JLR Papers in Press, May 16, 2005. DOI 10.1194/jlr.D500010-JLR200

¹ To whom correspondence should be addressed. e-mail: alex.brown{at}vanderbilt.edu

The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP₂/PIP₃) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP₂ compounds as well as 8 PIP₃ compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP₃ species and permitted us to propose a novel means for regulation and specificity in signaling through PIP₃.

This is the first reported identification of intact, cellular PIP₃ by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate.

Abbreviations: AfCS, Alliance for Cellular Signaling; LPA, lysophosphatidic acid; MCF, macrophage colony-stimulating factor; PIP, phosphatidylinositol monophosphate; PIP₂, phosphatidylinositol bisphosphate; PIP₃, phosphatidylinositol trisphosphate

Supplementary key words lipidomics • phosphoinositides • electrospray ionization mass spectrometry

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