Stable Isotope Dilution-Based Accurate Comparative Quantification of Nitrogen-Containing Metabolites inArabidopsis thalianaT87 Cells Usingin Vivo15N-Isotope Enrichment
- 1 January 2005
- journal article
- research article
- Published by Oxford University Press (OUP) in Bioscience, Biotechnology, and Biochemistry
- Vol. 69 (7) , 1331-1340
- https://doi.org/10.1271/bbb.69.1331
Abstract
Stable isotope dilution-based comparative quantification of nitrogen-containing metabolites for highly sensitive and selective metabolomics was developed using liquid chromatography/mass spectrometry (LC/MS) and 15N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with 15N-labeled medium. We found that the growth of cells maintained in 15N-labeled medium is very similar to the growth in normal medium, as evidenced by cell morphology, doubling time, and measurement of chlorophyll and carotenoid contents. Complete incorporation of 15N in folate, S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in T87 cells was accomplished after culturing for 21 d. Accurate comparative quantification of folate, SAM, and SAH was established by means of LC/MS using the isotopomers of the target metabolites as internal standards. The within- and between-run assay coefficients of variation for the folate, SAM, and SAH levels were all less than 8.5%. Stable isoto...Keywords
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