查看文章

A novel approach to ¹³C metabolic flux analysis (MFA) is presented using cytosolic metabolite pool sizes and their ¹³C labeling data from an isotopically non-stationary ¹³C labeling experiment (INST-CLE). The procedure is demonstrated with an E. coli wild type strain grown at fed batch conditions. The intra cellular labeling dynamics are excited by a sudden step increase of the ¹³C portion in the substrate feed. Due to unchanged saturation of the substrate uptake system, the metabolic fluxes remain constant during the following sampling time period of only 16s, in which 20 samples are taken by an automated rapid sampling device immediately stopping metabolism by methanol quenching. Subsequent cell disruptive sample preparation and LC–MS/MS enabled simultaneous determination of pool sizes and mass isotopomers of intra cellular metabolites requiring detection limits in the nM range. Based on this …