Metabolic flux analysis: Difference between revisions
Sunggyunglee (talk | contribs) Elaborated lead and included links to other Wiki articles. Also added image of flux map |
Sunggyunglee (talk | contribs) Added section to explain experimental component for MFA |
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[[File:Major metabolic fluxes in neuron-astrocyte coupling for resting conditions.png|thumb|350x350px|Example of metabolic flux map for metabolic pathways of astrocytes and neurons.]] |
[[File:Major metabolic fluxes in neuron-astrocyte coupling for resting conditions.png|thumb|350x350px|Example of metabolic flux map for metabolic pathways of astrocytes and neurons.]] |
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Metabolic flux analysis may use [[Carbon-13|<sup>13</sup>C]]-labeled isotope tracers for [[isotopic labeling]] experiments. Nuclear magnetic resonance ([[Nuclear magnetic resonance|NMR]]) techniques and [[mass spectrometry]] may then be used to measure metabolite labeling patterns to provide information for determination of pathway fluxes.<ref>{{Cite journal|last=Zamboni|first=Nicola|last2=Fendt|first2=Sarah-Maria|last3=Rühl|first3=Martin|last4=Sauer|first4=Uwe|date=2009-06-21|title=13C-based metabolic flux analysis|url=http://www.nature.com/articles/nprot.2009.58|journal=Nature Protocols|language=en|volume=4|issue=6|pages=878–892|doi=10.1038/nprot.2009.58|issn=1754-2189}}</ref> <ref name=":02" /><ref>{{Cite journal|last=Zamboni|first=Nicola|date=2011-02-01|title=13C metabolic flux analysis in complex systems|url=https://linkinghub.elsevier.com/retrieve/pii/S0958166910001394|journal=Current Opinion in Biotechnology|language=en|volume=22|issue=1|pages=103–108|doi=10.1016/j.copbio.2010.08.009}}</ref> Because MFA typically requires rigorous flux calculation of complex metabolic networks, publicly available software tools have been developed to automate MFA and reduce its computational burden. |
Metabolic flux analysis may use [[Carbon-13|<sup>13</sup>C]]-labeled isotope tracers for [[isotopic labeling]] experiments. Nuclear magnetic resonance ([[Nuclear magnetic resonance|NMR]]) techniques and [[mass spectrometry]] may then be used to measure metabolite labeling patterns to provide information for determination of pathway fluxes.<ref>{{Cite journal|last=Zamboni|first=Nicola|last2=Fendt|first2=Sarah-Maria|last3=Rühl|first3=Martin|last4=Sauer|first4=Uwe|date=2009-06-21|title=13C-based metabolic flux analysis|url=http://www.nature.com/articles/nprot.2009.58|journal=Nature Protocols|language=en|volume=4|issue=6|pages=878–892|doi=10.1038/nprot.2009.58|issn=1754-2189}}</ref> <ref name=":02" /><ref>{{Cite journal|last=Zamboni|first=Nicola|date=2011-02-01|title=13C metabolic flux analysis in complex systems|url=https://linkinghub.elsevier.com/retrieve/pii/S0958166910001394|journal=Current Opinion in Biotechnology|language=en|volume=22|issue=1|pages=103–108|doi=10.1016/j.copbio.2010.08.009}}</ref> Because MFA typically requires rigorous flux calculation of complex metabolic networks, publicly available software tools have been developed to automate MFA and reduce its computational burden. |
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== Experimental Method == |
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=== Isotope labeling experiments === |
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[[File:Workflow_of_isotope_labeling_experiment.pdf|thumb|320x320px|Simplified workflow of an example isotope labeling experiment. The black circle in the glucose tracer represents a labeled carbon atom, while the blue atoms represent an unlabeled carbon atom.]] |
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Isotope labeling experiments are optimal for gathering experimental data necessary for MFA. Because fluxes determine the isotopic labeling patterns of intracellular metabolites, measuring these patterns allows for inference of fluxes.<ref>{{Cite journal|last=Heuillet|first=Maud|last2=Bellvert|first2=Floriant|last3=Cahoreau|first3=Edern|last4=Letisse|first4=Fabien|last5=Millard|first5=Pierre|last6=Portais|first6=Jean-Charles|date=2018-02-06|title=Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments|url=https://doi.org/10.1021/acs.analchem.7b03886|journal=Analytical Chemistry|volume=90|issue=3|pages=1852–1860|doi=10.1021/acs.analchem.7b03886|issn=0003-2700}}</ref> The first step in the workflow of isotope labeling experiments is cell culture on labeled substrates. A substrate such as glucose is labeled by isotope(s), most often <sup>13</sup>C, and is introduced into the culture medium. The medium also typically contains vitamins and essential amino acids to facilitate cells' growth.<ref name=":3">{{Citation|last=Feng|first=Xueyang|title=Metabolic Pathway Determination and Flux Analysis in Nonmodel Microorganisms Through 13C-Isotope Labeling|date=2012|url=https://doi.org/10.1007/978-1-61779-827-6_11|work=Microbial Systems Biology: Methods and Protocols|pages=309–330|editor-last=Navid|editor-first=Ali|series=Methods in Molecular Biology|place=Totowa, NJ|publisher=Humana Press|language=en|doi=10.1007/978-1-61779-827-6_11|isbn=978-1-61779-827-6|access-date=2021-11-14|last2=Zhuang|first2=Wei-Qin|last3=Colletti|first3=Peter|last4=Tang|first4=Yinjie J.}}</ref> The labeled substrate is then metabolized by the cells, leading to the incorporation of the <sup>13</sup>C tracer in other intracellular metabolites. After the cells reach steady-state physiology (i.e., constant metabolite concentrations in culture), cells are then lysed to extract metabolites. For mammalian cells, extraction involves quenching of cells using [[methanol]] to stop their cellular metabolism and subsequent extraction of metabolites using methanol and water extraction.<ref>{{Cite journal|last=Sellick|first=Christopher A.|last2=Hansen|first2=Rasmus|last3=Stephens|first3=Gill M.|last4=Goodacre|first4=Royston|last5=Dickson|first5=Alan J.|date=2011-07-28|title=Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling|url=https://www.nature.com/articles/nprot.2011.366|journal=Nature Protocols|language=en|volume=6|issue=8|pages=1241–1249|doi=10.1038/nprot.2011.366|issn=1750-2799}}</ref> Concentrations of metabolites and labeled isotope in metabolites of the extracts are measured by instruments like [[Liquid chromatography–mass spectrometry|liquid chromatography-mass spectrometry]] or NMR, which also provide information on the position and number of labeled atoms on the metabolites.<ref name=":3" /> This data are necessary for gaining insight into the dynamics of intracellular metabolism and metabolite turnover rates to infer metabolic flux. |
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== Thermodynamics-Based Metabolic Flux Analysis<ref name=":1">{{Cite journal|last1=Hatzimanikatis|first1=Vassily|last2=Broadbelt|first2=Linda J.|author-link2=Linda Broadbelt|last3=Henry|first3=Christopher S.|date=2007-03-01|title=Thermodynamics-Based Metabolic Flux Analysis|journal=Biophysical Journal|language=en|volume=92|issue=5|pages=1792–1805|bibcode=2007BpJ....92.1792H|doi=10.1529/biophysj.106.093138|issn=0006-3495|pmc=1796839|pmid=17172310}}</ref> == |
== Thermodynamics-Based Metabolic Flux Analysis<ref name=":1">{{Cite journal|last1=Hatzimanikatis|first1=Vassily|last2=Broadbelt|first2=Linda J.|author-link2=Linda Broadbelt|last3=Henry|first3=Christopher S.|date=2007-03-01|title=Thermodynamics-Based Metabolic Flux Analysis|journal=Biophysical Journal|language=en|volume=92|issue=5|pages=1792–1805|bibcode=2007BpJ....92.1792H|doi=10.1529/biophysj.106.093138|issn=0006-3495|pmc=1796839|pmid=17172310}}</ref> == |
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Revision as of 09:53, 14 November 2021
Metabolic flux analysis (MFA) is an experimental fluxomics technique used to examine production and consumption rates of metabolites in a biological system. At an intracellular level, it allows for the quantification of metabolic fluxes, thereby elucidating the central metabolism of the cell.[1] Various methods of MFA, including isotopically stationary metabolic flux analysis, isotopically non-stationary metabolic flux analysis, and thermodynamics-based metabolic flux analysis, can be coupled with stoichiometric models of metabolism and mass spectrometry methods with isotopic mass resolution to elucidate the transfer of moieties containing isotopic tracers from one metabolite into another and derive information about the metabolic network. Metabolic flux analysis (MFA) has many applications such as determining the limits on the ability of a biological system to produce a biochemical such as ethanol,[2] predicting the response to gene knockout,[3][4] and guiding the identification of bottleneck enzymes in metabolic networks for metabolic engineering efforts.[5]
Metabolic flux analysis may use 13C-labeled isotope tracers for isotopic labeling experiments. Nuclear magnetic resonance (NMR) techniques and mass spectrometry may then be used to measure metabolite labeling patterns to provide information for determination of pathway fluxes.[6] [1][7] Because MFA typically requires rigorous flux calculation of complex metabolic networks, publicly available software tools have been developed to automate MFA and reduce its computational burden.
Experimental Method
Isotope labeling experiments
Isotope labeling experiments are optimal for gathering experimental data necessary for MFA. Because fluxes determine the isotopic labeling patterns of intracellular metabolites, measuring these patterns allows for inference of fluxes.[8] The first step in the workflow of isotope labeling experiments is cell culture on labeled substrates. A substrate such as glucose is labeled by isotope(s), most often 13C, and is introduced into the culture medium. The medium also typically contains vitamins and essential amino acids to facilitate cells' growth.[9] The labeled substrate is then metabolized by the cells, leading to the incorporation of the 13C tracer in other intracellular metabolites. After the cells reach steady-state physiology (i.e., constant metabolite concentrations in culture), cells are then lysed to extract metabolites. For mammalian cells, extraction involves quenching of cells using methanol to stop their cellular metabolism and subsequent extraction of metabolites using methanol and water extraction.[10] Concentrations of metabolites and labeled isotope in metabolites of the extracts are measured by instruments like liquid chromatography-mass spectrometry or NMR, which also provide information on the position and number of labeled atoms on the metabolites.[9] This data are necessary for gaining insight into the dynamics of intracellular metabolism and metabolite turnover rates to infer metabolic flux.
Thermodynamics-Based Metabolic Flux Analysis[11]
Thermodynamics-Based Metabolic Flux Analysis (TMFA) is a specialized type of metabolic flux analysis which utilizes linear thermodynamic constraints in addition to mass balance constraints to generate thermodynamically feasible fluxes and metabolite activity profiles. TMFA takes into consideration only pathways and fluxes that are feasible by using the Gibbs free energy change of the reactions and activities of the metabolites that are part of the model.[11]
See also
See Isotopic labeling for a brief treatment of stable isotope labeling.
References
- ^ a b Wiechert, Wolfgang (2001-07-01). "13C Metabolic Flux Analysis". Metabolic Engineering. 3 (3): 195–206. doi:10.1006/mben.2001.0187.
- ^ Papoutsakis, Eleftherios Terry; Meyer, Charles L. (1985-01-01). "Equations and calculations of product yields and preferred pathways for butanediol and mixed-acid fermentations". Biotechnology and Bioengineering. 27 (1): 50–66. doi:10.1002/bit.260270108. ISSN 0006-3592.
- ^ Burgard, Anthony P.; Maranas, Costas D. (2001-09-05). "Probing the performance limits of theEscherichia coli metabolic network subject to gene additions or deletions". Biotechnology and Bioengineering. 74 (5): 364–375. doi:10.1002/bit.1127. ISSN 0006-3592.
- ^ Henry, Christopher S.; Broadbelt, Linda J.; Hatzimanikatis, Vassily (2007-03-01). "Thermodynamics-Based Metabolic Flux Analysis". Biophysical Journal. 92 (5): 1792–1805. doi:10.1529/biophysj.106.093138. PMC 1796839. PMID 17172310.
{{cite journal}}: CS1 maint: PMC format (link) - ^ Antoniewicz, Maciek R. (2021-01-01). "A guide to metabolic flux analysis in metabolic engineering: Methods, tools and applications". Metabolic Engineering. Tools and Strategies of Metabolic Engineering. 63: 2–12. doi:10.1016/j.ymben.2020.11.002. ISSN 1096-7176.
- ^ Zamboni, Nicola; Fendt, Sarah-Maria; Rühl, Martin; Sauer, Uwe (2009-06-21). "13C-based metabolic flux analysis". Nature Protocols. 4 (6): 878–892. doi:10.1038/nprot.2009.58. ISSN 1754-2189.
- ^ Zamboni, Nicola (2011-02-01). "13C metabolic flux analysis in complex systems". Current Opinion in Biotechnology. 22 (1): 103–108. doi:10.1016/j.copbio.2010.08.009.
- ^ Heuillet, Maud; Bellvert, Floriant; Cahoreau, Edern; Letisse, Fabien; Millard, Pierre; Portais, Jean-Charles (2018-02-06). "Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments". Analytical Chemistry. 90 (3): 1852–1860. doi:10.1021/acs.analchem.7b03886. ISSN 0003-2700.
- ^ a b Feng, Xueyang; Zhuang, Wei-Qin; Colletti, Peter; Tang, Yinjie J. (2012), Navid, Ali (ed.), "Metabolic Pathway Determination and Flux Analysis in Nonmodel Microorganisms Through 13C-Isotope Labeling", Microbial Systems Biology: Methods and Protocols, Methods in Molecular Biology, Totowa, NJ: Humana Press, pp. 309–330, doi:10.1007/978-1-61779-827-6_11, ISBN 978-1-61779-827-6, retrieved 2021-11-14
- ^ Sellick, Christopher A.; Hansen, Rasmus; Stephens, Gill M.; Goodacre, Royston; Dickson, Alan J. (2011-07-28). "Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling". Nature Protocols. 6 (8): 1241–1249. doi:10.1038/nprot.2011.366. ISSN 1750-2799.
- ^ a b Hatzimanikatis, Vassily; Broadbelt, Linda J.; Henry, Christopher S. (2007-03-01). "Thermodynamics-Based Metabolic Flux Analysis". Biophysical Journal. 92 (5): 1792–1805. Bibcode:2007BpJ....92.1792H. doi:10.1529/biophysj.106.093138. ISSN 0006-3495. PMC 1796839. PMID 17172310.
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