Direct analysis of PI(3,4,5)P₃ using liquid chromatography electrospray ionization tandem mass spectrometry

Phosphatidylinositol (3,4,5) trisphosphate (PIP₃) is a biologically active membrane phospholipid that is essential for the growth and survival of all eukaryotic cells. We describe a new method that directly measures PIP₃ and describe the HPLC separation and measurement of the positional isomers of phosphatidylinositol bisphosphate, PI(3,5)P₂, PI(3,4)P₂ and PI(4,5)P₂. Mass spectrometric analyses were performed online using ultra-high performance liquid chromatography (UHPLC)-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the negative multiple-reaction monitoring (MRM) modes. Rapid separation of PIP₃ from PI, phosphatidylinositol phosphate (PIP) and PIP₂ was accomplished by C18 reverse phase chromatography with the addition of the ion pairing reagents diisopropylethanolamine (DiiPEA) and ethylenediamine tetraacetic acid tetrasodium salt dihydrate (EDTA) to the samples and mobile phase with a total run time, including equilibration, of 12 min. Importantly, these chromatography conditions result in no carryover of PIP, PIP₂, and PIP₃ between samples. To validate the new method, U87MG cancer cells were serum starved and treated with PDGF to stimulate PIP3 biosynthesis in the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Results generated with the LC/MS method were in excellent agreement with results generated using [³³P] phosphate radiolabeled U87MG cells and anion exchange chromatography analysis, a well validated method for measuring PIP₃. To demonstrate the usefulness of the new method, we generated reproducible IC₅₀ data for several well-characterized PI3K small molecule inhibitors using a U87MG cell-based assay as well as showing PIP₃ can be measured from additional cancer cell lines. Together, our results demonstrate this novel method is sensitive, reproducible and can be used to directly measure PIP₃ without radiolabeling or complex lipid derivatization.