DataEntryID 54 General information Manuscript title: Quantification of intracellular metabolites in Escherichia coli K12 using liquid chromatographic-electrospray ionization tandem mass spectrometric techniques. PubMed ID: http://www.ncbi.nlm.nih.gov/pubmed/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 Organism: Escherichia coli Strain: K-12 DSM Data type: metabolites at steady-state Data units: mM Execution date: not specified Experimental details Temperature (°C): not specified pH: not specified Carbon source: glucose Culture mode: chemostat Process condition: aerobic Dilution rate (h⁻¹): 0.125 Working volume: 0.4 L 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: 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 list: methanol-water; Analysis list: LC-ESI-MS; Methods description: 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. ------------References------------ [1] Skoog, D. A., and Leary, J. J. (1992) Principles of Instrumental Analysis, Saunders, New York. Data file: http://kimosys.org/repository/54/download?parameter=1138; Alternative formats: http://kimosys.org/repository/54/attached_files/130/download; http://kimosys.org/repository/54/attached_files/31/download; Submission and curation Entered by: Administrator KiMoSys Created: 2013-05-29 15:18:00 UTC Updated: 2020-04-24 16:10:32 UTC Version: 0 Status: (reviewed) 2013-12-06 17:17:38 UTC Views: 374 Downloads: 74