Metabolite Extraction Using Cold Solvent

The filter cultures were grown to OD₆₅₀ of 0.35, at which point metabolism was quenched and cells extracted by dropping the filters directly into 2.5 mL of −20°C 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid (acid is useful to ensure rapid and complete protein denaturation28 (link)). After 15 min, filters were washed with an additional 1 mL of extraction solvent. The combined extract was neutralized with ammonium hydroxide to avoid acid-catalyzed metabolite degradation. For quantification of metabolites from cells grown on U-¹³C-glucose, metabolite standards were added to the initial 2.5 mL of extraction solution, but not the subsequent 1 mL. For other experiments (quantification of glycerol and acetate cells, determination of unlabelled fraction, and determination of metabolite excretion), standard was not added to either solution.