Development of emerging methods in mass spectrometry based metabolomics

The presented project aims to break new technological ground in two current topics of metabolomics, i.e. high-throughput metabolic profiling of cell culture supernatants, and, in a second step, metabolic flux analysis of intracellular metabolites. The first part of the project addresses the optimization of existing methods of metabolic profiling methodology regarding throughput and compatibility with complex sample matrices, i.e. cell culture supernatants. The requirements for this analytical method are highly demanding and need fully independent strategies for development and validation compared to existing methods. The matrix represented by supernatants is very difficult, as various nutrients are present in ionic or neutral form at extremely high concentrations in combination with high molecular weight additives. This leads to incompatibility with so far developed and applied protocols ideal for e.g. ethanolic cell extracts and needs further development and validation of clean-up steps. Mass spectrometry based determination of isotopologue or tandem mass isotopomer fractions of intracellular metabolites for 13C-metabolic flux analysis (13C-MFA) represents an emerging research topic in the field of industrial biotechnology. However, the comprehensive determination of the metabolic fluxes in a biological system demands for the development of efficient chromatographic separations and optimum mass spectrometric detection strategies. Based on our developments for isotopologue analysis of proteinogenic amino acids, we plan to develop fit-for-purpose methods in project Part 2 meeting these requirements. The results of project part 1 will directly support these developments. However, as the concentration of intracellular metabolites is much lower and the amount of target compounds is much higher as in the latter example, the new methods have to provide more sensitivity and selectivity. Especially the determination of mass isotopomer fractions and distribution needs completely different measurement strategies, as a high number of transitions have to be acquired in mass spectrometric single reaction monitoring.