Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach.

Nature Protocols
Bryson D BennettJoshua D Rabinowitz

Abstract

This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with (13)C-labeled carbon sources, with detection of (13)C-assimilation by liquid chromatography-tandem MS. It enables absolute quantitation of several dozen metabolites over approximately 1 week of work.

References

Nov 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·S D Wilson, D W Horne
Feb 11, 2003·Analytical Biochemistry·Ram Prasad Maharjan, Thomas Ferenci
Mar 23, 2004·Analytical Biochemistry·Christoph WittmannElmar Heinzle
Feb 1, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Ian D WilsonEva M Lenz
Apr 20, 2005·Journal of Mass Spectrometry : JMS·Qizhi HuR Graham Cooks
Oct 22, 2005·Yeast·Silas G Villas-BôasJens Nielsen
Dec 15, 2005·Journal of the American Society for Mass Spectrometry·Wenyun LuJoshua D Rabinowitz
Aug 29, 2006·Nature Chemical Biology·Jie YuanJoshua D Rabinowitz
Sep 12, 2006·Analytical Biochemistry·Elizabeth Kimball, Joshua D Rabinowitz
Dec 13, 2006·Proceedings of the National Academy of Sciences of the United States of America·Matthew J BrauerJoshua D Rabinowitz
Feb 3, 2007·European Journal of Pharmaceutics and Biopharmaceutics : Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E.V·Davy GuillarmeJean-Luc Veuthey
Mar 16, 2007·Journal of the American Society for Mass Spectrometry·Wenyun LuJoshua D Rabinowitz
Mar 16, 2007·Proceedings of the National Academy of Sciences of the United States of America·Rhonda C MeyerThomas Altmann
Mar 23, 2007·Journal of Chromatography. a·Maria Rowena N Monton, Tomoyoshi Soga
Apr 12, 2007·Expert Review of Proteomics·Joshua D Rabinowitz
Jul 17, 2007·Analytical Chemistry·Joshua D Rabinowitz, Elizabeth Kimball
Sep 4, 2007·Journal of Chromatography. a·Dao T-T NguyenJean-Luc Veuthey

❮ Previous
Next ❯

Citations

Sep 27, 2012·Analytical and Bioanalytical Chemistry·C BueschlR Schuhmacher
Nov 13, 2010·Biotechnology Letters·Martin VolmerThomas Noll
Jun 30, 2009·Nature Chemical Biology·Bryson D BennettJoshua D Rabinowitz
Aug 21, 2008·Nature Protocols·Jie YuanJoshua D Rabinowitz
Jul 5, 2013·Nature Reviews. Cancer·Jason W Locasale
Nov 6, 2009·Molecular Biology of the Cell·Viktor M BoerJoshua D Rabinowitz
Jan 13, 2010·Briefings in Functional Genomics·Adrian D Hegeman
Sep 29, 2011·Applied and Environmental Microbiology·Daniel Amador-NoguezJoshua D Rabinowitz
Jan 29, 2011·Journal of Biomedicine & Biotechnology·Xueyang FengYinjie J Tang
May 11, 2012·Microbial Cell Factories·Alfred Fernández-CastanéJosep López-Santín
Dec 3, 2010·PloS One·Aleksandra Usenik, Matic Legiša
Nov 15, 2011·Bioanalysis·M E MerloR Breitling
Mar 20, 2014·Microbial Cell Factories·Arul M VarmanYinjie J Tang
Nov 13, 2013·Proceedings of the National Academy of Sciences of the United States of America·Mohona SarkarGary J Pielak
Mar 14, 2013·Journal of Microbiological Methods·Lei WangZongbao K Zhao
Aug 2, 2014·Bioresource Technology·Whitney D HollinsheadYinjie J Tang
Jan 15, 2016·Metabolomics : Official Journal of the Metabolomic Society·Shingo NoguchiEiichiro Fukusaki
Jul 16, 2014·Microbial Cell Factories·Ting ShiXueming Zhao
Mar 5, 2013·Molecular and Biochemical Parasitology·J Enrique Salcedo-Sora, Steve A Ward
Dec 29, 2011·Journal of Biotechnology·Alfred Fernández-CastanéJosep López-Santín
Jan 12, 2016·Clinical Proteomics·Christopher A CrutchfieldDaniel W Chan
Sep 18, 2015·MicrobiologyOpen·Samantha S SasnowLudmilla Aristilde
Feb 27, 2016·Journal of the American Chemical Society·Min XueJames R Heath

❮ 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.

Related Papers

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Wenyun LuJoshua D Rabinowitz
Expert Review of Proteomics
Joshua D Rabinowitz
© 2022 Meta ULC. All rights reserved