Fast sampling for quantitative microbial metabolomics: new aspects on cold methanol quenching: metabolite co-precipitation
The intra- and extracellular concentrations of 16 metabolites were measured in chemostat (D = 0.1 h−1) anaerobic cultures of the yeast Saccharomyces cerevisiae CEN.PK-113-7D growing on minimal medium. Two independent sampling workflows were employed: (i) conventional cold methanol quenching and (ii) a differential approach. Metabolites were quantified in different sample fractions (total, extracellular, quenching supernatant, methanol/water extract and pellet) in order to derive their mass balance. The differential method in combination with absolute metabolite quantification by gas-chromatography with isotope dilution mass spectrometry (GC–IDMS) was used as a benchmark to assess quality of the cold methanol quenching procedure. Quantitative comparison of metabolite concentrations in all fractions collected by different quenching techniques indicates asystematic loss of the total mass of various metabolites in course of the cold methanol quenching. Pellet resulting from the cold methanol quenching besides biomass contains considerable amounts of precipitated inorganic salts from the fermentation media. Quantitative analysis has revealed significant co-precipitation of polar extracellular metabolites together with these salts. This phenomenon is especially significant for metabolites with large extracellular mass-fraction. We report that the co-precipitation is a hitherto neglected phenomenon and concluded that its degree strongly linked to culturing conditions (i.e. media composition) and chemical properties of the particular metabolite. Thus, intracellular metabolite levels measured from samples collected by cold methanol quenching might be uncertain and variably biased due to corruption by described phenomena.
SEEK ID: https://fairdomhub.org/publications/244
DOI: 10.1007/s11306-014-0700-8
Projects: MOSES
Publication type: Not specified
Journal: Metabolomics
Citation: Metabolomics 11(2) : 286
Date Published: 1st Apr 2015
Registered Mode: Not specified
Authors: Maksim Zakhartsev, Oliver Vielhauer, Thomas Horn, Xuelian Yang, Matthias Reuss
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Created: 1st Nov 2015 at 17:50
Last updated: 8th Dec 2022 at 17:26
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Projects: PSYSMO, MOSES, COSMIC, BaCell-SysMO
Institutions: University of Stuttgart
Expertise: Reactor models, rapid sampling experiments, Systems Biology, Mathematical modeling, Regulatory Networks, Dynamics and Control of Biological Networks
Tools: Fermentation, stimulus response experiments, evaluation of process dynamics, continuous cultivation, Computational Systems Biology, including:- Dynamic modelling- Parameter estimation- Optimal experimental design- Dynamic optimization, Dynamic modelling, In silico Metabolic Network Analysis, fed-batch cultivation
Professor for Biochemcial Engineering, University Stuttgart
Projects: MOSES, ExtremoPharm, ZucAt, GenoSysFat, DigiSal, EraCoBiotech 2 nd call proposal preparation, FAIRDOM & LiSyM & de.NBI Data Structuring Training
Institutions: University of Stuttgart, University of Hohenheim, Norwegian University of Life Sciences, Norwegian University of Science and Technology
https://orcid.org/0000-0002-7973-9902Expertise: Biochemistry, coupling metabolome and environome, rapid sampling experiments, Systems Biology, carbon metabolism, Stoichiometric modelling, Proteomics, Metabolomics, yeast, fungi, Dynamics and Control of Biological Networks
Tools: Biochemistry and protein analysis, Metabolomics, Matlab, Fermentation, Chromatography, Material balance based modeling, stimulus response experiments, continuous cultivation, Enzyme assay, Mass spectrometry (LC-MS/MS), HPLC, GC and LC/MS analysis of metabolites, ODE, Parameter estimation
I've become a SysMO DB PAL for MOSES project in 2007 being a post-doc in lab of Prof. Matthias Reuss at University of Stuttgart. In the MOSES project, our major efforts were in the experimental data acquisition for dynamic model of primary carbon and anaerobic energy metabolism in yeast. The model implements prediction of perturbations of two types: glucose pulse and temperature jump. We implement “stimulus-response” methodology for the unraveling the dynamic structure of the network and to ...
SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".
The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.
Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...
Projects: BaCell-SysMO, COSMIC, SUMO, KOSMOBAC, SysMO-LAB, PSYSMO, SCaRAB, MOSES, TRANSLUCENT, STREAM, SulfoSys, SysMO DB, SysMO Funders, SilicoTryp, Noisy-Strep
Web page: http://sysmo.net/
MOSES (Micro Organism Systems biology: Energy and Saccharomyces cerevisiae) develops a new Systems Biology approach, which is called 'domino systems biology'. It uses this to unravel the role of cellular free energy ('ATP') in the control and regulation of cell function. MOSES operates though continuous iterations between partner groups through a new systems-biology driven data-management workflow. MOSES also tries to serve as a substrate for three or more other SYSMO programs.
Programme: SysMO
Public web page: http://www.moses.sys-bio.net/
Organisms: Saccharomyces cerevisiae
Investigation of dynamics of the central metabolism (glycolysis, PPP, anaplerotic reactions, purines) of yeast Saccharomyces cerevisiae in anaerobic conditions
Submitter: Maksim Zakhartsev
Studies: Metabolic perturbation of the steady state culture by glucose pulse
Assays: Biomass weight during glucose pulse, Cellular size and granularity during glucose pulse, Dynamics of extracellular metabolites during glucose pulse, Dynamics of intracellular metabolites during glucose pulse, Dynamics of macromolecules during glucose pulse, MOSES: dynamic model of glucose pulse
Snapshots: No snapshots
Steady state metabolic fluxes and metabolite concentrations of yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1
Submitter: Maksim Zakhartsev
Studies: Steady state concentrations of metabolites in yeast Saccharomyces cerevi..., Steady state fluxes in yeast Saccharomyces cerevisiae in anaerobic chemo...
Assays: Steady state concentrations of extracellular metabolites in yeast Saccha..., Steady state concentrations of intracellular metabolites in yeast Saccha..., Steady state extracellular fluxes in anaerobic yeast Saccharomyces cerev...
Snapshots: No snapshots
The steady state anaerobic culture (D = 0.1 h-1) was pertrubed by sudden increase of the extracellular glucose up to 1 g/L and both extra- and intracellular transient metabolite concentrations were measured
Submitter: Maksim Zakhartsev
Investigation: Kinetic analysis of metabolic system using tran...
Assays: Biomass weight during glucose pulse, Cellular size and granularity during glucose pulse, Dynamics of extracellular metabolites during glucose pulse, Dynamics of intracellular metabolites during glucose pulse, Dynamics of macromolecules during glucose pulse, MOSES: dynamic model of glucose pulse
Snapshots: No snapshots
Steady state fluxes in yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1
Submitter: Maksim Zakhartsev
Investigation: Steady state metabolic fluxes and metabolite co...
Assays: Steady state extracellular fluxes in anaerobic yeast Saccharomyces cerev...
Snapshots: No snapshots
Steady state concentrations of metabolites in yeast Saccharomyces cerevisiae in anaerobic chemostat at D=0.1 h-1
Submitter: Maksim Zakhartsev
Investigation: Steady state metabolic fluxes and metabolite co...
Assays: Steady state concentrations of extracellular metabolites in yeast Saccha..., Steady state concentrations of intracellular metabolites in yeast Saccha...
Snapshots: No snapshots
experimentally measured extracellular fluxes in yeast Saccharomyces cerevisiae in anaerobic glucose limited chemostat (D=0.1 h-1) on minimal medium
Submitter: Maksim Zakhartsev
Assay type: Metabolite Profiling
Technology type: HPLC
Investigation: Steady state metabolic fluxes and metabolite co...
Organisms: Saccharomyces cerevisiae
SOPs: Anaerobic media composition, Sampling of biomass, Yeast strains
Data files: Measured steady state metabolic fluxes
Snapshots: No snapshots
Steady state concentrations of extracellular metabolites in yeast Saccharomyces cerevisiae in anaerobic chemostat at D = 0.1 h-1 on minimal medium
Submitter: Maksim Zakhartsev
Assay type: Metabolite Profiling
Technology type: HPLC
Investigation: Steady state metabolic fluxes and metabolite co...
Organisms: Saccharomyces cerevisiae
SOPs: Anaerobic media composition, Sampling of biomass, Yeast strains
Data files: Measured steady state concentrations of extrace...
Snapshots: No snapshots
Submitter: Maksim Zakhartsev
Assay type: Metabolite Profiling
Technology type: Gas Chromatography Mass Spectrometry
Investigation: Steady state metabolic fluxes and metabolite co...
Organisms: Saccharomyces cerevisiae
SOPs: Anaerobic media composition, Determination of intracellular metabolites, Sampling of biomass, Yeast strains
Data files: Measured steady state concentrations of intrace...
Snapshots: No snapshots
Dynamics of intracellular metabolites (pyr, suc, fum, mal, akg, pep, g3p, 2pg, 3pg, cit, r5p, f6p, g6p, 6pg, ATP, ADP, AMP, UTP, GTP, inosine, NAD+, IMP, UDP, NADP+, CTP, AdenyloSuccinate, NADPH, trehalose) during glucose pulse. Glucose pulse was performed in anaerobically growing yeast Saccharomyces cerevisiae in steady state chemostat (D = 0.1 h-1) and transent concentrations of the extra- and intracellular metabolites from central carbon metabolism (e.g. glycolysis, PPP, glycerol, purines, ...
Creator: Maksim Zakhartsev
Submitter: Maksim Zakhartsev
Steady state concentrations of intracellular metabolites in yeast Saccharomyces cerevisiae anaerobic chemostat at D = 0.1 h-1 on minimal medium. All metabolite concentrations are in mmol/L(CV).
Creator: Maksim Zakhartsev
Submitter: Maksim Zakhartsev
Investigations: Kinetic analysis of metabolic system using tran..., Steady state metabolic fluxes and metabolite co...
Studies: Metabolic perturbation of the steady state cult..., Steady state concentrations of metabolites in y...
Assays: MOSES: dynamic model of glucose pulse, Steady state concentrations of intracellular me...
Steady state concentrations of extracellular metabolites in yeast Saccharomyces cerevisiae anaerobic chemostat at D = 0.1 h-1 on minimal medium. All metabolite concentrations are in mmol/L(R) except CO2, which is in parts of the partial pressure.
Creator: Maksim Zakhartsev
Submitter: Maksim Zakhartsev
Investigations: Kinetic analysis of metabolic system using tran..., Steady state metabolic fluxes and metabolite co...
Studies: Metabolic perturbation of the steady state cult..., Steady state concentrations of metabolites in y...
Assays: MOSES: dynamic model of glucose pulse, Steady state concentrations of extracellular me...
Steady state metabolic fluxes measured in glucose-limited chemostat of Saccharomyces cerevisiae at D = 0.1 h-1 growing on minimal medium. Fluxes are: glucose, ethanol, glycerol, acetate, succinate, pyruvate, lactate, citrate, malate, a-ketoglutarate, fumarate
Creator: Maksim Zakhartsev
Submitter: Maksim Zakhartsev
Investigations: Kinetic analysis of metabolic system using tran..., Steady state metabolic fluxes and metabolite co...
Studies: Metabolic perturbation of the steady state cult..., Steady state fluxes in yeast Saccharomyces cere...
Assays: MOSES: dynamic model of glucose pulse, Steady state extracellular fluxes in anaerobic ...
The model includes glycolysis, pentosephosphate pathway, purine salvage reactions, purine de novo synthesis, redox balance and biomass growth. The network balances adenylate pool as opened moiety.
Creator: Maksim Zakhartsev
Submitter: Maksim Zakhartsev
Model type: Metabolic network
Model format: SBML
Environment: Copasi
Organism: Saccharomyces cerevisiae
Investigations: Kinetic analysis of metabolic system using tran...