Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry

By methylating the phosphate groups of PtdIns(3,4,5)P 3 researchers can load this lipid more efficiently into a mass spectrometer and thus this lipid can be quantified in the presence of an internal synthetic standard. Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling 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 on 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 be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P 3 in unstimulated mouse and human cells (≥10 5 ) or tissues (≥0.1 mg) and their increase upon appropriate stimulation.