Improved 13C metabolic flux analysis in Escherichia coli metabolism: application of a high-resolution MS (GC–EI–QTOF) for comprehensive assessment of MS/MS fragments

Abstract

Abstract Gas chromatography–tandem mass spectrometry with electron ionization (GC–EI–MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC–EI–MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow that combines high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor–product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof–of–concept study, which confirmed the importance of the compiled library for high-resolution 13C-MFA. One sentence summary An analytical workflow that combines high-resolution mass spectrometry (MS) and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments, which provide positional information and therefore offering significant advantages over traditional MS to improve 13C-metabolic flux analysis.