(89d) Metabolic Flux Analysis Using 13C-Tracers and Tandem Mass Spectrometry

The Antoniewicz lab is pioneering the development of a new technique for metabolic flux analysis (MFA) based on stable-isotope labeling and tandem mass spectrometry (MS/MS). Here, we present the computational, analytical and experimental techniques we have developed for ¹³C-MFA using GC-MS/MS. As a practical example, we demonstrate for the first time the application of tandem MS for metabolic flux analysis in E. coli. We show that tandem MS offers considerable advantages for in vivo flux analysis compared to conventional mass spectrometry techniques (GC/MS and LC/MS). The number of independent measurements is greatly increased with tandem MS, as is the information content of the labeling measurements. As a result, metabolic fluxes are determined with much higher precision and accuracy. The application of tandem MS allowed us to quantify accurately metabolic pathway fluxes in E. coli that were previously unobservable with conventional MS and NMR techniques. In summary, our new tandem MS technique allows tracing of isotopic labeling in complex metabolic networks with much higher resolution and ultimately improves measurement of metabolic fluxes.