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General Information info

Manuscript title Sampling of intracellular metabolites for stationary and non-stationary 13C metabolic flux analysis in Escherichia coli
PubMed ID 25102204
Journal Analytical Biochemistry
Year 2014
Authors Pierre Millard, Stéphane Massou, Christoph wittmann, Jean-Charles Portais, Fabien Létisse
Affiliations 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
Keywords Quantitative metabolomics, Metabolism, Sampling procedures
Full text article Downloadarticle Millard_2014.pdf
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain K-12 MG1655
Data type metabolites at steady-state
Data units mM
Execution date not specified

Experimental Details info

Temperature (0C) 37.0
pH 7.0
Carbon source glucose,
Culture mode batch
Process condition aerobic
Dilution rate (h-1)
Working volume (L) 0.150
Biomass concentration (g/L)
Medium composition

Minimal synthetic medium: 5 mM KH2PO4, 10 mM Na2HPO4, 9 mM NaCl, 40 mM NH4Cl, 0.8 mM MgSO4, 0.1 mM CaCl2, 0.1 g/L thiamine, and 3 g/L glucose. Glucose and thiamine were sterilized by filtration (Minisart polyamide 0.2 μm, Sartorius, Göttingen, Germany), and other solutions were autoclaved separately.

General protocol information Sampling method: different methods (see Figure 1).

Quenching procedure: different methods (see Figure 1).

Extraction technique: hot ethanol

Sample analyzing method: NMR, GC-MS, LC-MS

Methods description - Notes

Samples were collected using the differential method described in detail in [1]. Briefly, 120 μl of broth or filtered extracellular medium (Sartolon polyamide 0.2 μm, Sartorius) was plunged with 120 μl of fully 13C-labeled cellular extract (used as internal standard) in 5 ml of an ethanol/water (75:25) solution at 95 °C, incubated for 2 min, cooled on ice, and stored at −80 °C. Samples were analyzed by ion chromatography (ICS 2500 system, Dionex, Sunnyvale, CA, USA) coupled with a 4000 QTrap triple quadrupole mass spectrometer (ABSciex, Framingham, MA, USA) equipped with a Turbo V source (ABSciex) for electrospray ionization [2]. The nebulizer gas pressure was 40 psi, the desolvation gas pressure was 50 psi, the desolvation gas temperature was 650 °C, and the capillary voltage was −3.3 kV. Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), 6-phosphogluconate (6PG), combined pools of xylulose-5-phosphate, ribose-5-phosphate, and ribulose-5-phosphate (P5P), sedoheptulose-7-phosphate (S7P), phosphoenolpyruvate (PEP), and combined pools of 2- and 3-phosphoglycerate (2/3PG) were analyzed in the multiple reaction monitoring (MRM) mode, and the isotope dilution mass spectrometry (IDMS) method [3] was used to ensure accurate quantification. Fragmentation was done by collision-activated dissociation using nitrogen as the collision gas at medium pressure. The daughter ion was a phosphate group (PO3−m/z = 79 or H2PO4−m/z = 97). Three samples of broth and filtrate were collected at the mid-exponential growth phase (OD600nm ∼ 1.2) and analyzed. From these data, we were able to quantify the relative fractions of intra- and extracellular metabolites in the total pools; we calculated absolute concentrations of intracellular metabolites assuming a cell volume of 1.77 ml/gDW [4].
--------------------------------------References------------------------------
[1] H. Taymaz-Nikerel, M. de Mey, C. Ras, A. ten Pierick, R.M. Seifar, J.C. van Dam, J.J. Heijnen, W.M. van Gulik, Anal. Biochem., 386 (2009), pp. 9–19. http://doi.org/bk5b8m
[2] P. Kiefer, C. Nicolas, F. Letisse, J.-C. Portais, Anal. Biochem., 360 (2007), pp. 182–188. http://doi.org/dws46h
[3] L. Wu, M.R. Mashego, J.C. van Dam, A.M. Proell, J.L. Vinke, C. Ras, W.A. van Windenn, W.M. van Gulik, J.J. Heijnen, Anal. Biochem., 336 (2005), pp. 164–171. http://doi.org/dp4h5k
[4] C. Chassagnole, N. Noisommit-Rizzi, J.W. Schmid, K. Mauch, M. Reuss, Biotechnol. Bioeng., 79 (2002), pp. 53–73. http://doi.org/dj2nzc

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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Homepage: http://kdbio.inesc-id.pt/kimosys
Interests: mathematical modeling, accessible data, use of data

Created 2015-05-01 11:55:03 UTC

Updated 2015-05-01 11:59:29 UTC

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Status (reviewed) 2015-05-01 12:03:29 UTC




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