What is the physiological significance of the fatty acyl chains of phosphoinositides. Phosphoinositides are a family of phospholipids that act as dynamic regulators of many important membrane events, for example signalling by cell surface receptors or trafficking between intracellular membranes (see Hawkins et al (2006) Biochem Soc Trans 34:647-62; Corvera et al (1999) Curr Opin Cell Biol 4:460-5 for background). We have recently developed a new method for measuring these lipids in biological samples using mass spectrometry (Clark et al (2011) Nat Methods 8:267-72). This method provides for the first time an accurate assessment of the fatty acyl composition of these lipids, particularly PIP2 and PIP3. Our preliminary data reveals that PIP2 and PIP3 molecular species are remarkably enriched in stearoyl and arachidonyl chains (in the SN1 and SN2 positions respectively) when samples are taken from animals (mice, rats and humans) but show much more diversity when analogous samples are examined from cells in culture. This suggests the fatty acyl composition of these lipids is highly regulated and is not reproduced in culture. This project will examine the effects of supplementing cells in culture with arachidonate and linoleic acids (the essential fatty acid that is thought to be the precursor of arachidonate in mammalian cells). It is hoped this approach will ‘recover’ the natural fatty acyl composition of PIP2 and PIP3 in cultured cells and lay the foundations for investigating its physiological significance, including a potential link between diet and intracellular signalling.