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Manuscript title Metabolic profiling of Escherichia coli cultivations: evaluation of extraction and metabolite analysis procedures.
PubMed ID 17479221
Journal Biotechnology Letters
Year 2007
Authors Julia Hiller, Ezequiel Franco-Lara, Dirk Weuster-Botz
Affiliations Institut fuer Bioverfahrenstechnik, Technische Universitaet Braunschweig, Gausstrasse 17, 38106 Braunschwieg, Germany
Keywords extraction methods, metabolic profiling and multi-substrate
Full text article no file uploaded
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain K-12 DSM 498
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 chemostat
Process condition aerobic
Dilution rate (h-1) 0.125
Working volume (L) 3.0
Biomass concentration (g/L) not specified
Medium composition

Defined medium: 30 g/l glucose, 0.2 g/l NH4Cl, 2g/l (NH4)2 SO4, 3.25 g/l KH2PO4, 2.5 g/l K2HPO4, 1.5 g/l NaH2PO4.H2O, 1 g/l, MgSO4.7H2O, 10 mg/l, CaCl2.2H2O, 0.5 mg/l, ZnSO47H2O, 0.25 mg/l CuCl2.2H2O, 2.5mg/l MnSO4.H2O, 1.75 mg/l CoCl2.6H2O, 0.125 mg/l H3BO3, 2.5 mg/l AlCl3.6H2O, 0.5mg/l Na2MoO4.2H2O, 18.3 mg/l, FeSO4.7H2O. Magnesium and trace-element solution.

General protocol information Sampling method: 6g cell suspension, 25 g 60% MeOH-H2O 30mM TEA, pH=7.5, T=40ºC

Quenching procedure: centrifugation at -19 ºC, 6000g, 3min

Extraction technique: freezing-thawing in methanol

Sample analyzing method: enzymatic, LC-MS

Methods description - Notes

Enzymatic assays - analyses of G6P, F6P, DHAP, GAP, FBP, PEP and PYR were performed by enzymatic assays according to Bergmeyer [1] modified for analysis with a microtiter plate photometer.
LC-MS analysis - analyses of AMP, cAMP, ADP, ATP, NAD, NADP, FAD and acetylcoenzymeA (AcCoA) were performed by an LC-MS method adopted from Buchholz et al. [2]. An Aquasil C18 column 250 x 4.6 mm2, 5 µm (Thermo, Dreieich, Germany) was applied for chromatography protected by a guard column of the same material. A degassed binary gradient at 0.3 ml min1 was achieved making use of a P 1100 HPLC pump (Thermo, Dreieich, Germany). Solvent A was 12 mM aqueous ammonium acetate, solvent B was methanol/solvent A (80:20, v/v). Equilibration time was 5 min (2% B). The gradient applied for separation was kept at 2% B for 5 min and increased to 100% B over the next 25 min. This level was held for 10 min. The injection volume was 20 µl, sample temperature was 4ºC and column temperature was set to 35ºC. HPLC flow was transferred directly to the mass spectrometer via the electro-spray ionisation (ESI) interface. ESI-MS analysis was performed using an LCQ Advantage iontrap mass spectrometer (Thermo Finnigan, Dreieich, Germany). N2 was used as sheath gas and helium served as damping gas. Data acquisition and analysis were conducted using the Xcalibur software. The following ESI parameters were employed: temperature of heated capillary: 350ºC; electrospray capillary voltage: 2.5 kV; sheath gas: 60 arbitrary units; auxiliary gas: 20 arbitrary units; detection of negative ions (50–850 u) in full scan mode.
See also figure 5 from the original article.

[1] Bergmeyer H (1985). Methods of enzymatic analysis, 3rd edn. Verlag Chemie, Weinheim.
[2] Buchholz A, Takors R, Wandrey C (2001). Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques. Anal Biochem 295:129–137.

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Entered by Administrator KiMoSysFirst name: Administrator
Affiliation: INESC-ID/IST
Interests: mathematical modeling, accessible data, use of data

Created 2013-05-23 12:57:59 UTC

Updated 2014-06-12 21:56:33 UTC

Version 0

Status (reviewed) 2013-12-06 17:17:38 UTC

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