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General Information info

Manuscript title Multiple High-Throughput Analyses Monitor the Response of E. coli to Perturbations.
PubMed ID 17379776
Journal Science
Year 2007
Authors Nobuyoshi Ishii, Kenji Nakahigashi, Tomoya Baba, Martin Robert, Tomoyoshi Soga, Akio Kanai, Takashi Hirasawa, Miki Naba, Kenta Hirai, Aminul Hoque, Pei Yee Ho, Yuji Kakazu, Kaori Sugawara, Saori Igarashi, Satoshi Harada, Takeshi Masuda, Naoyuki Sugiyama, Takashi Togashi, Miki Hasegawa, Yuki Takai, Katsuyuki Yugi, Kazuharu Arakawa, Nayuta Iwata, Yoshihiro Toya, Yoichi Nakayama, Takaaki Nishioka, Kazuyuki Shimizu, Hirotada Mori, Masaru Tomita
Affiliations Institute for Advanced Biosciences, Keio University
Keywords Escheirchia coli, perturbations response, chemostat, single knockouts
Full text article no file uploaded
Project name not specified

Experiment Description info

Organism Escherichia coli
Strain WT K-12 BW25113 and mutants
Data type enzyme/protein concentrations
Data units (mg-protein/g-dry cell weight)
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) 0.1, 0.2, 0.4, 0.5 and 0.7
Working volume (L) 1.0
Biomass concentration (g/L) See Supplementary files of the original article (Tabel S3).
Medium composition

Synthetic medium: 48mM Na2HPO4, 22mM KH2PO4, 10mM NaCl, 45mM (NH4)2SO4, 4g/L glucose, 1mM MgSO4, 1mg/L thiamin.HCL, 5.6 mg/L CaCl2, 8 mg/L FeCl3, 1 mg/L MnCl2.4H2O, 1.7 mg/L ZnCl2, 0.43 mg/L CuCl2.2H2O, 0.6 mg/L CoCl2.2H2O and 0.6 mg/L Na2MoO4.2H2O

General protocol information Measurement method: LC-MS/MS, 2D gel-MALDI-TOF

Methods description - Notes

Absolute protein quantification - to obtain absolute measurements of protein concentrations in complex samples we used a liquid chromatography / tandem mass spectrometry (LC-MS/MS) method that is a modification of previous work in absolute quantification making use of either external or internal standards with or without isotopically labelled synthetic peptides as standard [1-3]. The main characteristics of the current approach are that it combines mass spectrometric analysis in the multiple reaction monitoring mode (MRM), for specific and sensitive quantification of target proteins, with the use of peptides derived from purified recombinant proteins as external calibrants.
To first optimize the MS settings and select MRM parameters, LC-MS/MS analysis of Lys-C (Wako, Osaka, Japan) digests of each purified recombinant target protein was performed essentially as previously described [4]. Completion of digestion of both purified proteins and crude E. coli extracts was confirmed by measuring peptide yields with time and using different concentrations of Lys-C. LC-MS/MS analysis of peptides was performed on a QSTAR XL hybrid quadrupole time-of-flight instrument (AB/MDS-Sciex, Toronto, Canada) as previously described [4]. Different fragmentation voltage settings were tested to optimize marker peptide detection. Peptides that were readily observable (excluding N-terminal and C-terminal peptides which contain extra amino acids originating from the recombinant protein expression vector) and whose precursor and product ion pair was computationally found to be unique in the E. coli proteome were used as protein specific signals to quantify the corresponding endogenous protein target.
E. coli samples were cooled immediately on ice, washed twice by centrifugation with wash buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.0) and split into 1 ml aliquots. These were then centrifuged for 5 min at 5,000 g to collect cells, and pellets were stored at -80 ºC. To quantify target proteins in experimental E. coli samples, two tubes of frozen cell pellets collected from a single sampling of a continuous culture were used. Peptide mixtures derived from Lys-C digestion of recombinant standard proteins or from total E. coli cell extracts were similarly separated by LC-MS/MS on a 1.5 mm × 50 mm C18 reversed-phase column at a flow rate of 0.2 ml/min. Peptides were eluted with a twostep gradient combination (1% to 40% acetonitrile over 50 min and up to 50% over the next 5 min) and the column eluent electrosprayed into the API 3000 (AB/MDS-Sciex) triplequadrupole instrument for simultaneous monitoring of specific marker peptides for 68 different protein targets. MRM analysis was performed in positive mode with an ion-spray voltage of 5.5 kV and a monitoring time of 10 ms per peptide. To quantify proteins accurately, the peak areas in extracted ion current chromatograms corresponding to target specific peptides were integrated automatically using the Analyst software (ver.1.4.1, AB/MDS-Sciex), followed by manual verification, and the results were used to calculate the absolute amount of protein in the cell extracts. Absolute intracellular concentrations of proteins were calculated using the measured dry cell weight per 1 ml of culture.

[1] S. A. Gerber, J. Rush, O. Stemman, M. W. Kirschner, S. P. Gygi, Proc. Natl. Acad. Sci. U. S. A. 100, 6940 (2003).
[2] W. Wang et al., Anal. Chem. 75, 4818 (2003).
[3] L. Anderson, C. L. Hunter, Mol. Cell Proteomics 5, 573 (2006).
[4] K. Shinoda et al., J. Proteome Res. 5, 3312 (2006).

Data file
Downloadproteomic KIMODATAID44_v0.xlsx
Alternative format(s)
no file uploaded


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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 2013-05-03 14:04:25 UTC

Updated 2014-06-12 21:41:41 UTC

Version 0

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

Associated Models

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Model name Category Model Type Data used for Access Json
Authors: Ahmad A. Mannan, Yoshihiro Toya, Kazuyuki Shimizu, Johnjoe McFadden, Andrzej M. Kierzek , Andrea Rocco

Original paper: Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism.

kinetic model of the central carbon metabolism of E. coli Metabolism ordinary differential equations Model building Visto4 {"affiliation":"Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom ","article_file_name":null,"authors":"Ahmad A. Mannan, Yoshihiro Toya, Kazuyuki Shimizu, Johnjoe McFadden, Andrzej M. Kierzek , Andrea Rocco","biomodels_id":"","category":"Metabolism","combine_archive_content_type":null,"combine_archive_file_name":null,"combine_archive_file_size":null,"combine_archive_updated_at":null,"comments":"For the analysis and evaluation of the kinetic model, the solver ode15s was used.","control":null,"dilution_rate":"0.2","id":42,"journal":"Plos One","keywords":"E. coli, Bistability, Central Metabolism","main_organism":"Escherichia coli","manuscript_title":"Integrating Kinetic Model of E. coli with Genome Scale Metabolic Fluxes Overcomes Its Open System Problem and Reveals Bistability in Central Metabolism.","model_name":"kinetic model of the central carbon metabolism of E. coli ","model_type":"ordinary differential equations","organism_id":null,"project_name":"","pubmed_id":"26469081","review_journal_id":null,"sbml_file_name":"Mannan_2015.PDF","software":"MATLAB\u00ae R2007b (version 7.5.0)","used_for":"---\n- Model building\n","year":2015} Administrator KiMoSys

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