Quantification of PtdInsP(3) molecular species in cells and tissues by mass spectrometry

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signalling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based upon phosphate methylation coupled to high performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in un-stimulated murine and human cells (≥ 10(5)) or tissues (≥ 0.1 mg) and their increase upon appropriate stimulation.