Developing a Strategy for Stable Isotope Labelled Standards for Quantitation in Metabolomics and Systems Biology.

Metabolomics as a field is increasingly moving from qualitative measurements, whereby metabolic finger prints of a tissue or biofluid is used to categorize samples into groups, to quantitative measurements, particularly when used in systems biology. One common approach for quantitation in mass spectrometry is to use internal standards that are stably labelled with 13C, 15N or 2D, and hence can be distinguished by mass spectrometry but have near-identical chemical structures. The latter property ensures the same (or near similar) chromatography and ionisation of both the analyte and the standard (usually the kinetic isotope effect is small). However, a problem with this approach is the need to synthesize labelled metabolites which can be both time consuming and expensive, particularly if a mixture of metabolites are to be examined. Furthermore, there are relatively few sources of these labelled metabolites. The synthesis of labelled lipids is a major problem for the quantification of lipids, as there may be many hundreds of intact lipids in a typical tissue extract or blood plasma sample, and the different chemical classes of lipids may have markedly different ionisation efficiencies, necessitating the need for multiple standards. This PhD studentship will directly address this problem by exploring the use of stable isotope labelling in cultured yeast as part of a collaboration between the Griffin and Zhang/Oliver groups in the Department of Biochemistry, University of Cambridge and Selcia, a Small/Medium Enterprise (SME) specializing in the generation of labelled metabolites and drugs. The student will grow yeast on 13C labelled substrates under highly controlled fermentation conditions using state of the art facilities within the department's fermentation facility under the supervision of Dr Zhang and Prof. Oliver. We will then assess the use of the 13C labelled lipids synthesised in the yeast as internal standards for lipidomic studies in yeast, c.elegans and mice. This will include assessing the use of labelled glucose and amino acids during the culturing process. To assess the efficacy of our approach we will investigate strains with defined gene mutations associated with lipid metabolism (e.g. deletion of fatty acid desaturases) to investigate the impact of the gene deletion on the lipidome of the organism. Changes to the lipidome will be assessed using liquid chromatography mass spectrometry in conjunction with multivariate statistics. Such labelling approaches for the generation of internal standards in metabolomics have been used previously to derive labelled aqueous metabolites in E.coli but the use of yeast as a eukaryote should provide better representation of the labelled lipidome for other eukayotes, while still maintaining the ease of a microbial fermentation system. A key to our approach is to assess how reproducible the synthesis of lipids is by yeast under various conditions. For this we will apply a design of experiments (DOE) approach to understand what physical approaches most affect the synthesis of lipids in yeast. Using the analytical capabilities of Selcia we will also investigate the pre-fractionation of these lipids to provide a comprehensive and relatively cheap alternative to chemically synthesised lipids. This will provide training for the PhD studentship in chromatography and general analytical chemistry, with these skills highly sought in both academia and industry. In addition this PhD studentship will assess how such internal standards improve the analytical efficiency of metabolomic approaches as well as potentially developing a new source of stable isotopes for metabolomics, lipidomics and systems biology. There is the potential that if successful these internal standards could be marketed as part of the services offered by Selcia providing benefit both to Selcia and the University of Cambridge.