General Information info
|Manuscript title||Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.|
|Authors||Arne Buchholz, Ralf Takors, and Christian Wandrey|
|Affiliations||Institute of Biotechnology, Research Center Juelich, 52425 Juelich, Germany|
|Keywords||Escherichia coli, intracellular metabolites, ESI, extraction, LC-MS, quantification|
|Full text article||no file uploaded|
|Project name||not specified|
Experiment Description info
|Data type||metabolites at steady-state|
|Execution date||not specified|
Experimental Details info
|Temperature (0C)||not specified|
|Dilution rate (h-1)||0.125|
|Working volume (L)||0.4|
|Biomass concentration (g/L)||not specified|
Minimal medium: 30 g/L glucose, 0.2 g/L NH4Cl, 2.0 g/L (NH4)2SO4, 3.25 g/L KH2PO4, 2.5 g/L K2HPO4, 1.5 g/L NaH2PO4, 0.5 g/L MgSO4; trace substances: 1.0E-2 g/L CaCl2, 5.0E-4 g/L ZnSO4, 2.5E-4 CuCl2, 2.5E-3 g/L MnSO4, 1.75E-3 g/L CoCl2, 1.25E-4 g/L H3BO3, 2.5E-3 g/L AlCl3, 5.0E-4 g/L Na2MoO4, 1.0E-2 g/L FeSO4.
|General protocol information||
Samples are taken after the cultures have remained constant at a cell density of 10.0 g/L for at least three residence times, thus ensuring steady-state conditions.
Quenching procedure: Sampling was performed using precooled syringes containing 15 ml of the quenching fluid, 60%, v/v, aqueous methanol containing 70 mM Hepes (2-[4-(2-hydroxyethyl)- 1-piperazinyl]ethanesulfonic acid).
Extraction technique: methanol-water
Sample analyzing method: LC-ESI-MS
|Methods description - Notes||
Identification and Quantification of Metabolites - compounds were identified in cell extracts using retention time, m/z, specific fragmentation patterns, and spiking with known compounds. Quantification was accomplished via the [M-H]2 ion by applying the standard addition method . A standard solution containing all analytes at a known concentration was prepared. By spiking cell extracts with increasing amounts of this standard solution, linear regression plots of peak area versus concentration were obtained. At least three data points were averaged per concentration in a concentration range of 0.005–1.5 mM. Limits of detection (LOD) were determined by taking three times the standard error of the estimate for the regression of these linear regression plots.
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