Quenching and Extraction for Metabolomics

The quenching and extraction procedures for metabolome analysis and ¹³C-MFA were similar to the method described previously [49 (link)–51 (link)]. 10 mL culture was quenched in 5 mL 0.9% NaCl solution held at 0 °C in a liquid nitrogen bath. The mixtures were manually agitated to freeze the cells near 0 °C and centrifuged to remove the culture medium. To improve the precision of intracellular metabolite concentration measurements, we prepared uniformly ¹³C-labeled cell extract, which was used as internal standards (IS). The method was adapted from the studies of Wu et al. [52 (link)] and Weiner et al. [53 (link)] with slight modifications. For metabolite extraction, the cell pellets were suspended in 5 mL cold methanol containing IS. For ¹³C-MFA, IS was not added to the methanol. After sonication and vortexing with chloroform and Milli-Q water (72:28), the mixture was centrifuged for 5 min at 4600×g and 4 °C. The top layer was transferred into 5-kDa cutoff filters (Merck Millipore Ltd., Darmstadt, Germany) and then centrifuged to improve the extraction efficiency. The samples were completely lyophilized (FreeZone 6 Liter, Labconco, USA) and stored at − 80 °C.