Phosphoinositide profiling in complex lipid mixtures

Phosphoinositides (PIs, which are phosphorylated derivatives of phosphatidylinositol [PtdIns]) are a class of phospholipids concentrated at cytosolic leaflets of cellular membranes that function as major regulators of a wide variety of cellular processes. A unique feature of these phospholipids is that their hydrophilic inositol head group can be reversibly phosphorylated by lipid kinases and phosphatases, at the various hydroxyl positions, thus leading to the generation of seven naturally occurring isomeric species. Their differentially phosphorylated head groups function as ligands for cytoplasmic proteins as well as for cytosolic domains of membrane proteins and thus are main mediators of the signaling function of PIs. In addition, PIs serve as precursors of other intracellular second messengers. Their subcellular localization, which is spatially and temporally controlled, can be revealed in living cells by fluorescent fusion proteins of protein modules that bind specific stereoisomers. Commonly used methods for their detection in cell extracts include receptor-based assays and radiometric measurements of chromatographically separated products. These approaches report the position of the phosphate(s) on the inositol head group (isomer profiles), but are not easily applicable to large-scale studies and low-abundance samples. Recently, electrospray ionization mass spectrometry (ESI/MS) has been developed as a rapid and sensitive method to detect, identify, and quantify phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) with different fatty acid compositions (PI fatty acid profiles) in tissue and cell extracts. Here we review and discuss the current technologies available for PI profiling in complex lipid mixtures with an emphasis on chromatography and mass spectrometry.