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Title: High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry

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 » and accurate labeling information of intracellular AAs, and therefore a better definition of the fluxes.« less
Authors:
 [1] ;  [2] ;  [1]
  1. The Ohio State Univ., Columbus, OH (United States). Department of Molecular Genetics and Center for Applied Plant Sciences
  2. The Ohio State Univ., Columbus, OH (United States). Department of Molecular Genetics
Publication Date:
Grant/Contract Number:
SC0016490
Type:
Published Article
Journal Name:
Journal of Chromatography
Additional Journal Information:
Journal Volume: 1490; Journal Issue: C; Journal ID: ISSN 0021-9673
Publisher:
Elsevier
Research Org:
The Ohio State Univ., Columbus, OH (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
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
OSTI Identifier:
1379975
Alternate Identifier(s):
OSTI ID: 1429315

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. United States: N. p., 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. United States. doi:10.1016/j.chroma.2017.02.028.
Cocuron, Jean-Christophe, Tsogtbaatar, Enkhtuul, and Alonso, Ana P. 2017. "High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry". United States. doi: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 = {United States},
year = {2017},
month = {2}
}