RRC ID 4059
Author Kim JK, Harada K, Bamba T, Fukusaki E, Kobayashi A.
Title Stable isotope dilution-based accurate comparative quantification of nitrogen-containing metabolites in Arabidopsis thaliana T87 cells using in vivo (15)N-isotope enrichment.
Journal Biosci Biotechnol Biochem
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 (15)N-isotope enrichment. We produced metabolically stable isotope-labeled Arabidopsis T87 cells by culturing with (15)N-labeled medium. We found that the growth of cells maintained in (15)N-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 (15)N 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 isotope labeling by nitrogen source in Arabidopsis T87 cell culture provided simple, inexpensive, and accurate amino acid profiling. This interesting new protocol is valuable for the study of dynamic changes in N-compound pools in cultured cells.
Volume 69(7)
Pages 1331-40
Published 2005-7-1
DOI 10.1271/bbb.69.1331
PII JST.JSTAGE/bbb/69.1331
PMID 16041139
MeSH Arabidopsis / chemistry* Arabidopsis / cytology Chromatography, Liquid / methods Folic Acid / analysis Indicator Dilution Techniques / standards Mass Spectrometry / methods Nitrogen / analysis* Nitrogen / metabolism Nitrogen Isotopes Reproducibility of Results S-Adenosylhomocysteine / analysis S-Adenosylmethionine / analysis
IF 1.516
Times Cited 39
WOS Category CHEMISTRY, APPLIED FOOD SCIENCE & TECHNOLOGY BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Arabidopsis / Cultured plant cells, genes rpc00008