Metabolite extraction for mass spectrometry

To prepare samples for metabolite profiling, cells were washed once with ice-cold saline solution and 1 ml of 80% methanol added to each sample before vortexing and snap freezing in liquid nitrogen. Samples were subjected to three freeze-thaw cycles and centrifuged at 14,000 rpm for 15 min at 4°C. The metabolite-containing supernatant was evaporated until dry. Metabolites were reconstituted in 50 μl of 0.03% formic acid, vortex-mixed, and centrifuged to remove debris.