کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5135690 1493436 2017 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
High-throughput quantification of the levels and labeling abundance of free amino acids by liquid chromatography tandem mass spectrometry
چکیده انگلیسی


- Highly sensitive LC-MS/MS method quantifies free amino acids in the femtomol range.
- Sample processing is completely devoid of chemical derivatization.
- The mass isotopomer distribution of 19 free amino acids can be determined.

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.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography A - Volume 1490, 24 March 2017, Pages 148-155
نویسندگان
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