General Information info
|Manuscript title||Sampling of intracellular metabolites for stationary and non-stationary 13C metabolic flux analysis in Escherichia coli|
|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||Millard_2014.pdf|
|Project name||not specified|
Experiment Description info
|Data type||metabolites at steady-state|
|Execution date||not specified|
Experimental Details info
|Dilution rate (h-1)||—|
|Working volume (L)||0.150|
|Biomass concentration (g/L)||—|
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||
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 . 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 . 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  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 .
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