Reversed-Phase LC/MS Method for Polyphosphoinositide Analyses: Changes in Molecular Species Levels during Epidermal Growth Factor Activation in A431 Cells

In studies on lipid metabolomics, liquid chromatography/mass spectrometry (LC/MS) is a robust and popular technique. Although effective reversed-phase (RP) LC/MS methods enabling the separation of phospholipid molecular species have been developed, RPLC methods to analyze phosphatidylinositol phosphates (PIPs) have not been reported. In this study, we developed conditions suitable for PIP analysis. Coupled with (diethylamino)etbyl (DEAE)-cellulose pretreatment, at least 1 pmol each of phosphatidylinositol monophosphates (PIP I), bisphosphates (PIP2), and triphosphates standards per similar to 6 x 10(6) cultured cells could be measured. Using these methods, we detected elevated concentrations of more than 12 PIP1 species in epidermal growth factor (EGF)stimulated A431 cells, a human epidermoid carcinoma cell line. The PIP2 species detected were not elevated after stimulation. We also detected EGF-induced increases in the levels of several phosphatidic acid species using another set of methods. Our method sensitively determined PIPs within a biological sample and is thus suitable for analysis of phoisphoiniositide metabolism.