We present a novel approach utilizing stable isotope labeling and mass spectrometric detection, which we term differential kinetic flux profiling, to probe dynamic cellular metabolic changes in response to environmental perturbations. Cells are switched into isotope-labeled media both shortly before and separately shortly after the perturbation, and the kinetics of subsequent labeling of intracellular metabolites measured by liquid chromatography-tandem mass spectrometry. The combined data enable measurement of both metabolite formation from macromolecular decomposition and small molecule flux changes triggered by the environmental perturbation. This approach is exemplified using 15N-ammonia labeling of Escherichia coli to probe changes in amino acid metabolism induced by carbon starvation. We find that carbon starvation almost fully halts amino acid fluxes, and that the trace remaining flux is almost completely driven by protein degradation. The general approach of differential kinetic flux profiling should be of wide utility for dissecting cellular metabolic responses to changing environmental conditions.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry