What does this strategy enable?
Kinetic flux profiling follows replacement of unlabeled metabolites after switching a nutrient source to its isotope-labeled equivalent. The original demonstration used nitrogen assimilation in cultured E. coli; the transferable contribution is the link between labeling dynamics, pool sizes and flux.
Transferable workflow
Workflow
- 1
Switch nutrient isotope labeling
Change the isotopic nutrient source while preserving the intended culture conditions.
- 2
Sample the labeling time course
Quench and extract replicate samples at times that resolve metabolite turnover.
- 3
Measure labeling and pool sizes
Use chromatography-MS to quantify unlabeled fractions and intracellular metabolite concentrations.
- 4
Fit the specified kinetic model
Estimate turnover and flux using equations appropriate to precursor labeling and pathway structure.
Evidence of reuse
Published implementations and adaptations
These papers applied, adapted, extended, benchmarked or formalized the Strategy after its originating publication. They are examples of precedent, not a ranking of software choices.
Kinetic flux profiling for quantitation of cellular metabolic fluxes
Expanded experimental and quantitative procedure, including culture handling and kinetic interpretation.
Strategy profile
Related objectives
Stable-isotope tracing
Components
Sample preparation, Data acquisition, Statistical analysis
Scope and considerations
Designed for
- Flux estimation in cultured cells with experimentally resolved labeling kinetics.
Important requirements
- Rapid nutrient switching, timed quenching, isotope-resolved measurements and pool-size quantification.
Considerations
- Basic interpretation assumes metabolic steady state; dynamic variants require additional modeling.
- Reliable quantification requires resolving turnover relative to upstream precursor labeling.
- A single endpoint or labeling half-time without pool-size and model context does not establish absolute flux.
Reusable resources
Publication
Yuan et al. Kinetic flux profiling of nitrogen assimilation in Escherichia coli. Nature Chemical Biology (2006).