Kinetic flux profiling for quantitation of cellular metabolic fluxes.

Nature Protocols
Jie YuanJoshua D Rabinowitz

Abstract

This protocol enables quantitation of metabolic fluxes in cultured cells. Measurements are based on the kinetics of cellular incorporation of stable isotope from nutrient into downstream metabolites. At multiple time points, after cells are rapidly switched from unlabeled to isotope-labeled nutrient, metabolism is quenched, metabolites are extracted and the extract is analyzed by chromatography-mass spectrometry. Resulting plots of unlabeled compound versus time follow variants of exponential decay, with the flux equal to the decay rate multiplied by the intracellular metabolite concentration. Because labeling is typically fast (t(1/2)<or=5 min for central metabolites in Escherichia coli), variations on this approach can effectively probe dynamically changing metabolic fluxes. This protocol is exemplified using E. coli and nitrogen labeling, for which quantitative flux data for approximately 15 metabolites can be obtained over 3 d of work. Applications to adherent mammalian cells are also discussed.

References

Apr 1, 1970·The Biochemical Journal·D W TempestC M Brown
May 10, 2002·Environmental Microbiology·Jeremy S EdwardsBernhard Palsson
Sep 5, 2002·Biotechnology and Bioengineering·Diana VisserJoseph J Heijnen
Nov 5, 2002·Proceedings of the National Academy of Sciences of the United States of America·Daniel SegrèGeorge M Church
Feb 27, 2003·European Journal of Biochemistry·Eliane Fischer, Uwe Sauer
May 6, 2003·Annual Review of Microbiology·Larry Reitzer
Oct 22, 2005·Yeast·Silas G Villas-BôasJens Nielsen
Aug 29, 2006·Nature Chemical Biology·Jie YuanJoshua D Rabinowitz
Sep 12, 2006·Analytical Biochemistry·Elizabeth Kimball, Joshua D Rabinowitz
Oct 7, 2006·Biotechnology Progress·Jochen SchaubMichael Dauner
Nov 15, 2006·Molecular Systems Biology·Uwe Sauer
Dec 2, 2006·Biotechnology Progress·Jin-Woo Kim, Tonya L Peeples
Dec 13, 2006·Proceedings of the National Academy of Sciences of the United States of America·Matthew J BrauerJoshua D Rabinowitz
Feb 3, 2007·Proceedings of the National Academy of Sciences of the United States of America·Natalie C DuarteBernhard Ø Palsson
Apr 12, 2007·Expert Review of Proteomics·Joshua D Rabinowitz
Jul 10, 2007·Journal of the American Chemical Society·Jie Yuan, Joshua D Rabinowitz
Jul 17, 2007·Analytical Chemistry·Joshua D Rabinowitz, Elizabeth Kimball
Sep 4, 2007·Analytical Biochemistry·Mariët J van der WerfThomas Hankemeier
Dec 19, 2007·NMR in Biomedicine·Cristina CudalbuDanielle Graveron-Demilly
May 27, 2008·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Wenyun LuJoshua D Rabinowitz
Jul 19, 2008·Biotechnology and Bioengineering·K SchmidtJ Villadsen
Aug 21, 2008·Nature Protocols·Bryson D BennettJoshua D Rabinowitz

❮ Previous
Next ❯

Citations

Jul 7, 2011·Applied Microbiology and Biotechnology·Katharina Nöh, Wolfgang Wiechert
Oct 19, 2011·Analytical Chemistry·Jurre J KamphorstJoshua D Rabinowitz
Jun 30, 2009·Nature Chemical Biology·Bryson D BennettJoshua D Rabinowitz
Feb 9, 2012·Proceedings of the National Academy of Sciences of the United States of America·Laura J TerryThomas Shenk
May 8, 2013·Proceedings of the National Academy of Sciences of the United States of America·Jason VoSydney Kustu
Feb 28, 2013·The Plant Cell·Marek SzecowkaStéphanie Arrivault
Sep 29, 2011·Applied and Environmental Microbiology·Daniel Amador-NoguezJoshua D Rabinowitz
Jan 29, 2011·Journal of Biomedicine & Biotechnology·Xueyang FengYinjie J Tang
Sep 27, 2012·BMC Systems Biology·David ByrneDaniel Segrè
Mar 14, 2012·BMC Systems Biology·Sabrina Kleessen, Zoran Nikoloski
Feb 1, 2012·BMC Systems Biology·Jan SchellenbergerBernhard Palsson
Oct 16, 2013·Proceedings of the National Academy of Sciences of the United States of America·Jianhai DuJames B Hurley
Jul 6, 2014·Nature Protocols·Robert HeiseAlisdair R Fernie
May 13, 2014·Current Opinion in Biotechnology·Kevin ChoGary J Patti
Apr 24, 2012·Nature Chemical Biology·Yi-Fan XuJoshua D Rabinowitz
Mar 5, 2013·Nature Chemical Biology·Olesya A UlanovskayaBenjamin F Cravatt
Feb 16, 2016·Cell Reports·Candice Sun HongHeather R Christofk
Mar 10, 2016·Progress in Nuclear Magnetic Resonance Spectroscopy·Teresa W-M Fan, Andrew N Lane
Dec 5, 2014·Proceedings of the National Academy of Sciences of the United States of America·Stefanie Renee DeVitoJoshua Munger
Dec 26, 2015·Metabolic Engineering·Lisa Maria DerschChristoph Wittmann
Nov 28, 2015·Current Opinion in Biotechnology·Doug K Allen
Jan 2, 2016·Current Opinion in Biotechnology·Hai Shi, Jörg Schwender
Mar 4, 2015·Current Opinion in Biotechnology·Joerg M BuescherSarah-Maria Fendt
Dec 23, 2014·Current Opinion in Biotechnology·Sebastian NiedenführKatharina Nöh
Mar 5, 2013·Molecular and Biochemical Parasitology·J Enrique Salcedo-Sora, Steve A Ward
Jan 4, 2012·Pharmacology & Therapeutics·Teresa W-M FanAndrew N Lane
Jun 15, 2011·Trends in Microbiology·Catherine L WinderRoyston Goodacre
Sep 14, 2010·Current Opinion in Biotechnology·Nicola Zamboni
Jul 6, 2010·Drug Discovery Today·Miroslava Cuperlović-CulfIan Chute
May 1, 2010·Current Opinion in Microbiology·Natsumi SaitoMasaru Tomita

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

© 2022 Meta ULC. All rights reserved