Sample preparation related to the intracellular metabolome of yeast methods for quenching, extraction, and metabolite quantitation

The determination of intracellular metabolite concentrations in Saccharomyces cerevisiae cell systems requires appropriate experimental methods to (a) collect cells and rapidly inhibit metabolism (quenching), (b) fracture cell walls and extract metabolites from within the cellular envelope(s), and (c) detect and quantify metabolites. A range of methods are applied for each of these processes, and no single method is appropriate for all metabolites. For example, the physicochemical diversity of metabolites, including solubility in water or organic solvents, is large. No single extraction solvent is appropriate for all metabolites reported in S. cerevisiae, and multiple solvent systems for extraction employing water, methanol, and chloroform at different pH are recommended for targeted extraction of metabolites. In this chapter, methods for the targeted study of organic acids present in the tricarboxylic acid cycle will be described. These include (a) the quenching of metabolism in batch cell cultures, (b) a single extraction method which provides the extraction of a wide diversity of metabolites, and (c) an analytical method applying gas chromatography-mass spectrometry for targeted analysis of six organic acids present in the tricarboxylic acid cycle metabolic pathway.