The synthesis of tools for the quantification by mass spectroscopy of biologically relevant Phosphatidyl Inositol Phosphates

There are a number of lipid (fat) molecules known collectively as Phosphatidyl Inositol Phosphates (PIPs) that are important components in the communication of events within cells. When these molecules are not found at appropriate levels, the communication pathways within the cells do not work properly and this leads to changes which can be catastrophic to the normal functioning of the cell, and can lead to a wide range of effects from cell death to uncontrollable cell growth. PIPs are made from an inositol 'head' which can have phosphates added to it, a glycerol unit and one or two fatty acid chains added to the glycerol unit. Traditionally it has only been possible to study the position of phosphate groups on the inositol part of PIPs, however, at the Babraham Institute we have developed mass spectroscopy methods which allow us, for the first time, to look both at changes in the phosphates and differences in the fatty acid composition in intact PIPs. What we have found is that changes in both the fatty acid composition of the PIPs and levels of phosphorylation occur in malfunctioning cells. This work is in the early stages and we do not yet understand why these changes occur or what they mean. To further the development of this important method we require synthetic standards of the biologically active PIPs that we have identified and which cannot be obtained commercially. These standards will be used to calibrate the mass spectrometer so that we can accurately measure the levels of PIPs in tissue samples. These standards will also confirm that we have assigned the structures correctly by the comparison of the mass spectroscopic data and will allow us to extend our analyses to further biologically important PIP species. The development of new methods to accurately measure PIPs will help both academics and the pharmaceutical industry develop novel therapies aimed at regulating the levels of these phospholipids in cells. Indeed, almost every large pharmaceutical company in the world currently has programmes aimed at inhibiting the production of at least one of these PIP species and hence these methods will have immediate applicability in supporting this effort. Further, the possibility of measuring the fatty acyl composition of PIPs may open up a whole new area of research examining the impact of dietary fatty acids on the function of these molecules.

Within the BI Inositide Laboratory we have developed a sensitive technique to measure intact PIPs by mass spectrometry. This reveals both the fatty acid side chain composition and the degree of phosphorylation of the inositol head group. To progress this work further we now require relevant standards, both labelled (13C) and unlabelled to allow structural confirmation and accurate quantitative data to be obtained. We have extensive experience of synthesising PIPs, based on methods previously developed by our collaborator Andrew Holmes in the University of Cambridge Dept of Chemistry (Holmes et al J.Chem.Soc.PerkinTrans.I,1999,923-935). Some modification will be required in the later stages of the synthesis, but these are trivial steps so that the synthesis is compatible with the presence of double bonds in the FA chains. We have already prepared a number of saturated analogues with P(4,5) and P(3,4,5) head groups and used these as internal standards and tools to develop the mass spectroscopic method thus far. We can now detect naturally occurring PIP, PIP2 and PIP3s in standard biological preparations, including growth factor-stimulated cell lines and tumour biopsies.Through this work we have realised that closely related PIPn species (same head group and very similar fatty acyl chains) undergo unpredictable loses during LC/MS, necessitating the use of appropriate standards to make truly quantitative measurements. The measurement of PIP3 is likely sufficient as a measure of the PI3K signalling pathway in cells (since the P(3,4,5) head is the only one found in cells) but it will important to extend our analyses to positional isomers of PIP2 and PIP, since these species characterise the activity of different pathways (eg PI3P versus PI4P or PI34P2 verses PI45P2). Our favoured approach to achieving this is to develop new HPLC methods to separate PIPs prior to MS. The standards we plan to create will be invaluable in selecting appropriate solvent and column systems

There is a significant research effort within the pharmaceutical industry to identify new treatments in oncology and inflammation by targeting phosphatidyl inositol phosphate signalling pathways, in particular the PI3K signalling pathway. The development of a new, widely applicable, quantitative assay of PIP3 in cell and tissue samples would make a highly significant contribution to this research effort. It would allow the pharmaceutical industry to directly assay the activity of the PI3K pathway in clinical samples and would allow a direct assessment of the effects of their PI3K inhibitors at various stages of the drug discovery process. Currently, poorly quantitative assessments of individual protein effectors of this pathway of contentious validity (eg PKB phosphorylation) are used for this purpose. The ability to look properly at changes in the fatty acid (FA) composition of phosphatidylinositol phosphates could open up a whole new field investigating the effects of diet, age and health on these important biological molecules.