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

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

doi:10.1016/j.chroma.2017.02.028

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

Journal Article · Wed Mar 01 00:00:00 EST 2017 · Journal of Chromatography

DOI:https://doi.org/10.1016/j.chroma.2017.02.028· OSTI ID:1379975

Cocuron, Jean-Christophe; Tsogtbaatar, Enkhtuul; Alonso, Ana P.

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.

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Research Organization:: The Ohio State Univ., Columbus, OH (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0016490

OSTI ID:: 1379975

Alternate ID(s):: OSTI ID: 1429315; OSTI ID: 1657861

Journal Information:: Journal of Chromatography, Journal Name: Journal of Chromatography Vol. 1490 Journal Issue: C; ISSN 0021-9673

Publisher:: ElsevierCopyright Statement

Country of Publication:: Netherlands

Language:: English

Citation Metrics:

Cited by: 21 works

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

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