Phosphoinositides remain a key interest in the UK.
Lipid research has a long history in Europe. In the past, it has progressed in two distinct research communities: the signalers, and the metabolic and membrane lipid biologists. However, the advent of lipidomic methodologies, in particular advances in liquid chromatography-mass spectrometry, has helped to bring these communities together.
In the UK, there is a nascent group called UKLipidomics that has brought together the main UK lipidomics labs, the bioinformaticians and others who wish to benefit from the power of lipidomic analysis. In Europe as a whole, 26 labs from 14 countries are part of the European Commission FP7-funded LipidomicNet large-scale integrating project focusing on lipid droplets. In contrast to the U.S. LIPID Metabolites and Pathways Strategy effort, the European program is less focused on method development and more centered on translational research. However, there is regular collaboration between some of the European and the U.S. groups and, in particular, there is a clear emphasis on ensuring commonality in data analysis, bioinformatics and nomenclature. The inclusion of Fritz Spener, Gerrit van Meer and myself on the International Lipid Classification and Nomenclature Committee together with LIPID MAPS colleagues and Takao Shimizu and Masahiro Nishijima from Japan has gone a long way toward achieving this. However, the lipid community needs to ensure that this communication and collaboration continues as the methodologies become more widespread.
Much of the lipid research in the UK remains heavily focused on signaling, and phosphoinositides are a particular emphasis. PI-3-kinase research has matured to the point where inhibitors are being trialed extensively. Nevertheless major questions remain, particularly relating to the physiological roles of individual isoforms and the importance of distinct molecular species of PtdIns(3,4,5)P3. The development of knock-out and knock-in mice is facilitating the former analysis while the development of novel mass spectrometry-based analysis is assisting in addressing the latter.
The analysis of PtdIns(3,4,5)P3 by mass spectrometry has been a persistent problem in the lipidomics field. A number of methods have been developed, including an earlier procedure from my lab, but these have proven, for a number of reasons, to be unreliable and thus have not been adopted widely. One of the major reasons for this has been the very nature of the phosphate headgroups that provide the lipid with its signaling properties. These charged molecules bind strongly to glass, steel and proteins, and thus their recovery, not just from cells but from analytical systems, can be poor. Additionally, the phosphates can be labile and migrate around the inositol ring when exposed to the extreme pH sometimes used to extract the lipid.
A recent publication from our institute has reported a solution to this problem (1). The novel methodology involves methylation of the phosphate groups and the use of neutral loss mass spectrometry following reverse phase chromatography to identify molecular species of PtdIns(3,4,5)P3, PtdInsP2 and PtdInsP. As yet, the procedure cannot separate the different PtdInsP2 and PtdInsP forms, but use of the method allows the quantitative determination of PtdIns(3,4,5)P3 species in cells and tissues. The technique will be of great use in both research studies and in monitoring clinical trials. Intriguingly, we find that distinct molecular species of PtdIns(3,4,5)P3 are produced in cells, confirming earlier work from Alex Brown (2) and supporting the concept of specificity of individual lipid molecular species rather than classes in cell function.
1. Clark, J., Anderson, K. E., Juvin, V., Smith, T. S., Karpe, F., Wakelam, M. J. O., Stephens, L. R., and Hawkins, P. T. (2011) Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry. Nat. Methods 8, 267-272.
2. Milne, S. B., Ivanova, P. T., DeCamp, D., Hsueh, R. C., and Brown, H. A. (2005) A targeted mass spectrometric analysis of phosphatidylinositol phosphate species. J. Lipid Res. 46, 1796 – 1802.
Michael Wakelam (firstname.lastname@example.org) is director of the Babraham Institute in Cambridge, UK.