Abstract
Metabolic flux, the flow of metabolites through networks of enzymes, represents the dynamic productive output of cells. Improved understanding of intracellular metabolic fluxes will enable targeted manipulation of metabolic pathways of medical and industrial importance to a greater degree than is currently possible. Flux balance analysis (FBA) is a constraint-based approach to modeling metabolic fluxes, but its utility is limited by a lack of experimental measurements. Incorporation of experimentally measured fluxes as system constraints will significantly improve the overall accuracy of FBA. We applied a novel, two-tiered approach in the yeast Saccharomyces cerevisiae to measure nutrient consumption rates (extracellular fluxes) and a targeted intracellular flux using a 14C-labeled precursor with HPLC separation and flux quantitation by accelerator mass spectrometry (AMS). The use of AMS to trace the intracellular fate of 14C-glutamine allowed the calculation of intracellular metabolic flux through this pathway, with glutathione as the metabolic end point. Measured flux values provided global constraints for the yeast FBA model which reduced model uncertainty by more than 20%, proving the importance of additional constraints in improving the accuracy of model predictions and demonstrating the use of AMS to measure intracellular metabolic fluxes. Our results highlight the need to use intracellular fluxes to constrain the models. We show that inclusion of just one such measurement alone can reduce the average variability of model predicted fluxes by 10%.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 9812-9817 |
| Number of pages | 6 |
| Journal | Analytical Chemistry |
| Volume | 82 |
| Issue number | 23 |
| DOIs | |
| State | Published - Dec 1 2010 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Analytical Chemistry
Cite this
Yeast dynamic metabolic flux measurement in nutrient-rich media by hplc and accelerator mass spectrometry. / Stewart, Benjamin J.; Navid, Ali; Turteltaub, Ken W; Bench, Graham.
In: Analytical Chemistry, Vol. 82, No. 23, 01.12.2010, p. 9812-9817.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Yeast dynamic metabolic flux measurement in nutrient-rich media by hplc and accelerator mass spectrometry
AU - Stewart, Benjamin J.
AU - Navid, Ali
AU - Turteltaub, Ken W
AU - Bench, Graham
PY - 2010/12/1
Y1 - 2010/12/1
N2 - Metabolic flux, the flow of metabolites through networks of enzymes, represents the dynamic productive output of cells. Improved understanding of intracellular metabolic fluxes will enable targeted manipulation of metabolic pathways of medical and industrial importance to a greater degree than is currently possible. Flux balance analysis (FBA) is a constraint-based approach to modeling metabolic fluxes, but its utility is limited by a lack of experimental measurements. Incorporation of experimentally measured fluxes as system constraints will significantly improve the overall accuracy of FBA. We applied a novel, two-tiered approach in the yeast Saccharomyces cerevisiae to measure nutrient consumption rates (extracellular fluxes) and a targeted intracellular flux using a 14C-labeled precursor with HPLC separation and flux quantitation by accelerator mass spectrometry (AMS). The use of AMS to trace the intracellular fate of 14C-glutamine allowed the calculation of intracellular metabolic flux through this pathway, with glutathione as the metabolic end point. Measured flux values provided global constraints for the yeast FBA model which reduced model uncertainty by more than 20%, proving the importance of additional constraints in improving the accuracy of model predictions and demonstrating the use of AMS to measure intracellular metabolic fluxes. Our results highlight the need to use intracellular fluxes to constrain the models. We show that inclusion of just one such measurement alone can reduce the average variability of model predicted fluxes by 10%.
AB - Metabolic flux, the flow of metabolites through networks of enzymes, represents the dynamic productive output of cells. Improved understanding of intracellular metabolic fluxes will enable targeted manipulation of metabolic pathways of medical and industrial importance to a greater degree than is currently possible. Flux balance analysis (FBA) is a constraint-based approach to modeling metabolic fluxes, but its utility is limited by a lack of experimental measurements. Incorporation of experimentally measured fluxes as system constraints will significantly improve the overall accuracy of FBA. We applied a novel, two-tiered approach in the yeast Saccharomyces cerevisiae to measure nutrient consumption rates (extracellular fluxes) and a targeted intracellular flux using a 14C-labeled precursor with HPLC separation and flux quantitation by accelerator mass spectrometry (AMS). The use of AMS to trace the intracellular fate of 14C-glutamine allowed the calculation of intracellular metabolic flux through this pathway, with glutathione as the metabolic end point. Measured flux values provided global constraints for the yeast FBA model which reduced model uncertainty by more than 20%, proving the importance of additional constraints in improving the accuracy of model predictions and demonstrating the use of AMS to measure intracellular metabolic fluxes. Our results highlight the need to use intracellular fluxes to constrain the models. We show that inclusion of just one such measurement alone can reduce the average variability of model predicted fluxes by 10%.
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UR - http://www.scopus.com/inward/citedby.url?scp=78649696098&partnerID=8YFLogxK
U2 - 10.1021/ac102065f
DO - 10.1021/ac102065f
M3 - Article
C2 - 21062031
AN - SCOPUS:78649696098
VL - 82
SP - 9812
EP - 9817
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 23
ER -