Format

Send to

Choose Destination
Anal Biochem. 2014 Nov 15;465:38-49. doi: 10.1016/j.ab.2014.07.026. Epub 2014 Aug 4.

Sampling of intracellular metabolites for stationary and non-stationary (13)C metabolic flux analysis in Escherichia coli.

Author information

1
Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR 5504, F-31400 Toulouse, France.
2
Universität des Saarlande, Systembiotechnologie Campus, D-66123 Saarbrücken, Germany.
3
Université de Toulouse, INSA, UPS, INP, LISBP, F-31077 Toulouse, France; INRA, UMR 792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR 5504, F-31400 Toulouse, France. Electronic address: fabien.letisse@insa-toulouse.fr.

Abstract

The analysis of metabolic intermediates is a rich source of isotopic information for (13)C metabolic flux analysis ((13)C-MFA) and extends the range of its applications. The sampling of labeled metabolic intermediates is particularly important to obtain reliable isotopic information. The assessment of the different sampling procedures commonly used to generate such data, therefore, is crucial. In this work, we thoroughly evaluated several sampling procedures for stationary and non-stationary (13)C-MFA using Escherichia coli. We first analyzed the efficiency of these procedures for quenching metabolism and found that procedures based on cold or boiling solvents are reliable, in contrast to fast filtration, which is not. We also showed that separating the cells from the broth is not necessary in isotopic stationary state conditions. On the other hand, we demonstrated that the presence of metabolic intermediates outside the cells strongly affects the transient isotopic data monitored during non-stationary (13)C-labeling experiments. Meaningful isotopic data can be obtained by recovering intracellular labeled metabolites from pellets of cells centrifuged in cold solvent. We showed that if the intracellular pools are not separated from the extracellular ones, accurate flux maps can be established provided that the contribution of exogenous compounds is taken into account in the metabolic flux model.

KEYWORDS:

(13)C-labeling experiments; Isotopes; Isotopologue; Mass isotopomer; Metabolism; Sampling procedures

PMID:
25102204
DOI:
10.1016/j.ab.2014.07.026
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center