High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry
Abstract
Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reaction monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more completemore »
- Authors:
- Publication Date:
- Research Org.:
- The Ohio State Univ., Columbus, OH (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1379975
- Alternate Identifier(s):
- OSTI ID: 1429315; OSTI ID: 1657861
- Grant/Contract Number:
- SC0016490
- Resource Type:
- Published Article
- Journal Name:
- Journal of Chromatography
- Additional Journal Information:
- Journal Name: Journal of Chromatography Journal Volume: 1490 Journal Issue: C; Journal ID: ISSN 0021-9673
- Publisher:
- Elsevier
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Amino acids; 13C-labeling; LC–MS/MS; Mass isotopomer distribution; Multiple reaction monitoring; Metabolic flux analysis; LC-MS/MS; Metabolic flux analysis.
Citation Formats
Cocuron, Jean-Christophe, Tsogtbaatar, Enkhtuul, and Alonso, Ana P. High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry. Netherlands: N. p., 2017.
Web. doi:10.1016/j.chroma.2017.02.028.
Cocuron, Jean-Christophe, Tsogtbaatar, Enkhtuul, & Alonso, Ana P. High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry. Netherlands. https://doi.org/10.1016/j.chroma.2017.02.028
Cocuron, Jean-Christophe, Tsogtbaatar, Enkhtuul, and Alonso, Ana P. Wed .
"High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry". Netherlands. https://doi.org/10.1016/j.chroma.2017.02.028.
@article{osti_1379975,
title = {High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry},
author = {Cocuron, Jean-Christophe and Tsogtbaatar, Enkhtuul and Alonso, Ana P.},
abstractNote = {Accurate assessment of mass isotopomer distributions (MIDs) of intracellular metabolites, such as free amino acids (AAs), is crucial for quantifying in vivo fluxes. To date, the majority of studies that measured AA MIDs have relied on the analysis of proteinogenic rather than free AAs by: i) GC–MS, which involved cumbersome process of derivatization, or ii) NMR, which requires large quantities of biological sample. In this work, the development and validation of a high-throughput LC–MS/MS method allowing the quantification of the levels and labeling of free AAs is described. Sensitivity in the order of the femtomol was achieved using multiple reaction monitoring mode (MRM). The MIDs of all free AAs were assessed without the need of derivatization, and were validated (except for Trp) on a mixture of unlabeled AA standards. Finally, this method was applied to the determination of the 13C-labeling abundance in free AAs extracted from maize embryos cultured with 13C-glutamine or 13C-glucose. Although Cys was below the limit of detection in these biological samples, the MIDs of a total of 18 free AAs were successfully determined. Due to the increased application of tandem mass spectrometry for 13C-Metabolic Flux Analysis, this novel method will enable the assessment of more complete and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.},
doi = {10.1016/j.chroma.2017.02.028},
journal = {Journal of Chromatography},
number = C,
volume = 1490,
place = {Netherlands},
year = {Wed Mar 01 00:00:00 EST 2017},
month = {Wed Mar 01 00:00:00 EST 2017}
}
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Works referenced in this record:
Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry
journal, October 2000
- Petritis, K.; Chaimbault, P.; Elfakir, C.
- Journal of Chromatography A, Vol. 896, Issue 1-2
Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering
journal, January 2011
- Alonso, Ana P.; Val, Dale L.; Shachar-Hill, Yair
- Metabolic Engineering, Vol. 13, Issue 1
Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis
journal, April 2016
- Sá, João V.; Kleiderman, Susanne; Brito, Catarina
- Neurochemical Research, Vol. 42, Issue 1
Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos: Metabolic fluxes in sunflower embryos
journal, August 2007
- Alonso, Ana P.; Goffman, Fernando D.; Ohlrogge, John B.
- The Plant Journal, Vol. 52, Issue 2
Biosynthetically Directed Fractional 13C-labeling of Proteinogenic Amino Acids. An Efficient Analytical Tool to Investigate Intermediary Metabolism
journal, September 1995
- Szyperski, Thomas
- European Journal of Biochemistry, Vol. 232, Issue 2
Non-protein amino acids in plant defense against insect herbivores: Representative cases and opportunities for further functional analysis
journal, September 2011
- Huang, Tengfang; Jander, Georg; de Vos, Martin
- Phytochemistry, Vol. 72, Issue 13
The biosynthesis of plant alkaloids and nitrogenous microbial metabolitesThis review and those that preceded it are dedicated to my three teachers who were each a marvellous scientific inspiration: F. G. Holliman, A. R. Battersby and G. Stork. They were the giants onto whose shoulders I was privileged to climb.
journal, January 2003
- Herbert, Richard B.
- Natural Product Reports, Vol. 20, Issue 5
The origin and evolution of lignin biosynthesis: Tansley review
journal, June 2010
- Weng, Jing-Ke; Chapple, Clint
- New Phytologist, Vol. 187, Issue 2
Analysis of Proteinogenic Amino Acid and Starch Labeling by 2D NMR
book, October 2013
- Truong, Quyen; Shanks, Jacqueline V.
- Plant Metabolic Flux Analysis
Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS: Metabolic flux profiling in E.���coli
journal, March 2003
- Fischer, Eliane; Sauer, Uwe
- European Journal of Biochemistry, Vol. 270, Issue 5
LC−MS/MS Assay for Protein Amino Acids and Metabolically Related Compounds for Large-Scale Screening of Metabolic Phenotypes
journal, November 2007
- Gu, Liping; Jones, A. Daniel; Last, Robert L.
- Analytical Chemistry, Vol. 79, Issue 21
13C metabolic flux analysis in complex systems
journal, February 2011
- Zamboni, Nicola
- Current Opinion in Biotechnology, Vol. 22, Issue 1
Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS
journal, January 2007
- Iwatani, Shintaro; Van Dien, Stephen; Shimbo, Kazutaka
- Journal of Biotechnology, Vol. 128, Issue 1
Measuring Complete Isotopomer Distribution of Aspartate Using Gas Chromatography/Tandem Mass Spectrometry
journal, April 2012
- Choi, Jungik; Grossbach, Matthew T.; Antoniewicz, Maciek R.
- Analytical Chemistry, Vol. 84, Issue 10
Targeted Metabolomics of Physaria fendleri, an Industrial Crop Producing Hydroxy Fatty Acids
journal, February 2014
- Cocuron, Jean-Christophe; Anderson, Brooke; Boyd, Alison
- Plant and Cell Physiology, Vol. 55, Issue 3
Reliable Metabolic Flux Estimation in Escherichia coli Central Carbon Metabolism Using Intracellular Free Amino Acids
journal, May 2014
- Okahashi, Nobuyuki; Kajihata, Shuichi; Furusawa, Chikara
- Metabolites, Vol. 4, Issue 2
Fractionation of plant extracts using ion-exchange Sephadex
journal, September 1980
- Redgwell, R. J.
- Analytical Biochemistry, Vol. 107, Issue 1
Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments: Probing subcellular metabolism in seeds
journal, February 2012
- Allen, Doug K.; Laclair, Russell W.; Ohlrogge, John B.
- Plant, Cell & Environment, Vol. 35, Issue 7
Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry
journal, September 1999
- Chaimbault, P.; Petritis, K.; Elfakir, C.
- Journal of Chromatography A, Vol. 855, Issue 1
Understanding fatty acid synthesis in developing maize embryos using metabolic flux analysis
journal, September 2010
- Paula Alonso, Ana; Dale, Val L.; Shachar-Hill, Yair
- Metabolic Engineering, Vol. 12, Issue 5
Metabolic networks in motion: 13 C‐based flux analysis
journal, January 2006
- Sauer, Uwe
- Molecular Systems Biology, Vol. 2, Issue 1
Analysis of native amino acids by liquid chromatography/electrospray ionization mass spectrometry: comparative study between two sources and interfaces
journal, January 2008
- de Person, Marine; Chaimbault, Patrick; Elfakir, Claire
- Journal of Mass Spectrometry, Vol. 43, Issue 2
Highlighting the tricarboxylic acid cycle: Liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids
journal, May 2013
- Koubaa, Mohamed; Cocuron, Jean-Christophe; Thomasset, Brigitte
- Analytical Biochemistry, Vol. 436, Issue 2
Tyrosine hydroxylase and regulation of dopamine synthesis
journal, April 2011
- Daubner, S. Colette; Le, Tiffany; Wang, Shanzhi
- Archives of Biochemistry and Biophysics, Vol. 508, Issue 1
Publishing 13C metabolic flux analysis studies: A review and future perspectives
journal, November 2013
- Crown, Scott B.; Antoniewicz, Maciek R.
- Metabolic Engineering, Vol. 20
Isotopically nonstationary 13 C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation
journal, November 2014
- Ma, Fangfang; Jazmin, Lara J.; Young, Jamey D.
- Proceedings of the National Academy of Sciences, Vol. 111, Issue 47
Evaluating 13C enrichment data of free amino acids for precise metabolic flux analysis
journal, November 2011
- Mori, Eiji; Furusawa, Chikara; Kajihata, Shuichi
- Biotechnology Journal, Vol. 6, Issue 11
A roadmap for interpreting 13 C metabolite labeling patterns from cells
journal, August 2015
- Buescher, Joerg M.; Antoniewicz, Maciek R.; Boros, Laszlo G.
- Current Opinion in Biotechnology, Vol. 34
Plant Defense against Insect Herbivores
journal, May 2013
- Fürstenberg-Hägg, Joel; Zagrobelny, Mika; Bak, Søren
- International Journal of Molecular Sciences, Vol. 14, Issue 5
Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis
journal, July 2011
- Locasale, Jason W.; Grassian, Alexandra R.; Melman, Tamar
- Nature Genetics, Vol. 43, Issue 9
Biosynthesis, function and metabolic engineering of plant volatile organic compounds
journal, February 2013
- Dudareva, Natalia; Klempien, Antje; Muhlemann, Joëlle K.
- New Phytologist, Vol. 198, Issue 1
Synergy between 13C-metabolic flux analysis and flux balance analysis for understanding metabolic adaption to anaerobiosis in E. coli
journal, January 2011
- Chen, Xuewen; Alonso, Ana P.; Allen, Doug K.
- Metabolic Engineering, Vol. 13, Issue 1
Accurate Assessment of Amino Acid Mass Isotopomer Distributions for Metabolic Flux Analysis
journal, October 2007
- Antoniewicz, Maciek R.; Kelleher, Joanne K.; Stephanopoulos, Gregory
- Analytical Chemistry, Vol. 79, Issue 19
Amino Acid Biosynthesis and its Regulation
journal, June 1978
- Umbarger, H. E.
- Annual Review of Biochemistry, Vol. 47, Issue 1
NMR-based fluxomics: Quantitative 2D NMR methods for isotopomers analysis
journal, August 2007
- Massou, Stéphane; Nicolas, Cécile; Letisse, Fabien
- Phytochemistry, Vol. 68, Issue 16-18
Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts
journal, April 2005
- Blank, Lars M.; Lehmbeck, Frank; Sauer, Uwe
- FEMS Yeast Research, Vol. 5, Issue 6-7, p. 545-558
Ion-pair chromatography on a porous graphitic carbon stationary phase for the analysis of twenty underivatized protein amino acids
journal, February 2000
- Chaimbault, P.; Petritis, K.; Elfakir, C.
- Journal of Chromatography A, Vol. 870, Issue 1-2
Collisional fragmentation of central carbon metabolites in LC-MS/MS increases precision of 13C metabolic flux analysis
journal, October 2011
- Rühl, Martin; Rupp, Beat; Nöh, Katharina
- Biotechnology and Bioengineering, Vol. 109, Issue 3