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General Information info

Manuscript title Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques.
PubMed ID 11488613
Journal Analytical Biochemistry
Year 2001
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

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

Experimental Details info

Temperature (0C) not specified
pH not specified
Carbon source glucose,
Culture mode chemostat
Process condition aerobic
Dilution rate (h-1) 0.125
Working volume (L) 0.4
Biomass concentration (g/L) not specified
Medium composition

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 Sampling method: 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 [1]. 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.

[1] Skoog, D. A., and Leary, J. J. (1992) Principles of Instrumental Analysis, Saunders, New York.

Data file
Downloadmetabolites KIMODATAID54_v0.xlsx
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Submission and curation info

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

Created 2013-05-29 15:18:00 UTC

Updated 2014-05-02 22:21:40 UTC

Version 0

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

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