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
- 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