High-throughput quantitative metabolomics: workflow for cultivation, quenching, and analysis of yeast in a multiwell format.
Metabolomics is a founding pillar of quantitative biology and a valuable tool for studying metabolism and its regulation. Here we present a workflow for metabolomics in microplate format which affords high-throughput and yet quantitative monitoring of primary metabolism in microorganisms and in particular yeast. First, the most critical step of rapid sampling was adapted to a multiplex format by using fritted 96-well plates for cultivation, which ensure fast sample transfer and permit us to use well-established quenching in cold solvents. Second, extensive optimization of large-volume injection on a GC/TOF instrument provided the sensitivity necessary for robust quantification of 30 primary metabolites in 0.6 mg of yeast biomass. The metabolome profiles of baker's yeast cultivated in fritted well plates or in shake flasks were equivalent. Standard deviations of measured metabolites were between 10% and 50% within one plate. As a proof of principle we compared the metabolome of wild-type Saccharomyces cerevisiae and the single-deletion mutant Delta sdh1, which were clearly distinguishable by a 10-fold increase of the intracellular succinate concentration in the mutant. The described workflow allows the production of large amounts of metabolome samples within a day, is compatible with virtually all liquid extraction protocols, and paves the road to quantitative screens.
Ewald
Jennifer Christina
JC
Institute of Molecular Systems Biology, ETH Zurich, Wolfgang-Pauli Strasse 16, 8093 Zurich, Switzerland.
Heux
Stéphanie
S
Zamboni
Nicola
N