GC-MS/MS survey of collision-induced dissociation of tert-butyldimethylsilyl-derivatized amino acids and its application to 13C-metabolic flux analysis of Escherichia coli central metabolism
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Stable isotope labeling experiments using mass spectrometry have been employed to investigate carbon flow levels (metabolic flux) in mammalian, plant, and microbial cells. To achieve a more precise 13C-metabolic flux analysis (13C-MFA), novel fragmentations of tert-butyldimethylsilyl (TBDMS)-amino acids were investigated by gas chromatography-tandem mass spectrometry (GC-MS/MS). The product ion scan analyses of 15 TBDMS-amino acids revealed 24 novel fragment ions. The amino acid-derived carbons included in the five fragment ions were identified by the analyses of 13C-labeled authentic standards. The identification of the fragment ion at m/z 170 indicated that the isotopic abundance of S-methyl carbon in methionine could be determined from the cleavage of C5 in the precursor of [M–159]+ (m/z 218). It was also confirmed that the precision of 13C-MFA in Escherichia coli central carbon metabolism could be improved by introducing 13C-labeling data derived from novel fragmentations.
Keywords13C-metabolic flux analysis GC-MS/MS Fragmentation Amino acids Escherichia coli
We thank Prof. Eiichiro Fukusaki and Prof. Yoshihiro Toya (Osaka University, Japan) for their helpful comments. This research was partially supported by JST, Strategic International Collaborative Research Program, SICORP, for JP-US Metabolomics.
Compliance with ethical standards
All remaining authors have declared no conflicts of interest.
- 22.Toya Y, Ishii N, Nakahigashi K, Hirasawa T, Soga T, Tomita M. 13C-metabolic flux analysis for batch culture of Escherichia coli and its Pyk and Pgi gene knockout mutants based on mass isotopomer distribution of intracellular metabolites. Biotechnol Prog. 2010;26(4):975–92.Google Scholar