The development of a powerful new lipidomics tool kit underpinning our objectives

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Tools for lipidomic analysis of biological extracts have kept pace with their proteomic and DNA-sequencing counterparts. It is now possible to quantify the large majority of lipids and, where relevant, their families of molecular species (ie with different hydrocarbon chains); over 1000 in mammalian cells(126). However, some key problems remain. Most relevant to our ISP has been the difficulty in detecting and quantifying phosphoinositides, particularly the more polar species and their regio-isomers (eg resolving PI(3,4)P2 from PI(4,5)P2 and PI(3,5)P2) and their families of molecular species. This problem is most extreme for PI(3,4,5)P3 and PI(3,4)P2 despite huge interest in their central roles as the output signals from class I PI3Ks. Hence the only approaches that can quantify them cannot resolve molecular species and are low-through-put. This has had 2 major impacts on the field. Firstly, that there is very little data quantifying the primary lipid signals from this pathway PI(3,4)P2 and PIP3, instead many workers use surrogate readouts such as phosphorylation of PKB, and hence there are real problems in trying to understand the dynamics of the pathway as part of the process of developing models to describe its operating principles. Furthermore, it is very difficult to understand the roles and significance of a number of PIP3 and PI(3,4)P2 phosphatases in the pathway because of a lack of these types of measurement. Both of these problems are also hindering pharmaceutical activity in this area. Secondly, it left major conceptual questions about the significance of the distinct molecular species of phosphoinositides. Do they carry any of the PIP3 code? We have shown that chemically synthesized C16:0/C16:0-PIP3 (number of carbon:number of double bonds; dipalmitoyl) was an order of magnitude less potent than C18:0/C20:4-PIP3 (stereoyl/arachidonyl) in activation of PKB in vitro(31). Raising, although not validating, the plausibility of this hypothesis. We have developed a mass-spec-based methodology opening the application of lipidomic strategies to PIP3 and have shown that up to 7 distinct molecular species of PIP3 can be found in cells with a pattern of abundance similar to that of other phosphoinositides, with a predominance of stereoyl/arachidonyl-species(34). Importantly, however, we found that stereoyl/arachidonyl species were further preferentially concentrated in PIP3 compared to the PI(4,5)P2 that acted as the cellular substrate. This must be because the class I PI3Ks or the PIP3-phosphatases preferentially metabolise certain molecular species, either because of differences in the apparent rate constants towards, or access to, the different species. We have hypothesised this is because the molecular species may provide an additional element, “colour”, to the PIP3 code that might be associated with preferential targeting of the lipids into different micro-domains in the plasma membrane known to be enriched in specific proteins. This work is of direct significance to our final high-level objective.

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