title: Integrative normalization and comparative analysis for metabolic fingerprinting by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.
abstract: Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC-TOF-MS) was applied to the comparative metabolic fingerprinting of a wild-type versus a double mutant strain of Escherichia coli lacking the transhydrogenases UdhA and PntAB. Using peak lists generated with the Leco ChromaTOF software as input, we developed retention time correction and data alignment tools (INCA). The accuracy of peak alignment and detection of 1.1- to 4-fold changes in metabolite concentration was validated by a spike-in experiment with 20 standard compounds. A list of 48 significant features that differentiated the two E. coli strains was obtained with an estimated false discovery rate (FDR) of <0.05. A total of 27 metabolites, mainly from the citrate cycle, were identified. That the signal intensity of the m/z 73 trace of the trimethylsilyl (TMS) group reflected true differences in metabolite abundance was confirmed by quantification of pyruvate, fumarate, malate, succinate, alpha-ketoglutarate, citrate, cis-aconitate, myo-inositol, and glucose-6-phosphate using compound specific fragment ions and stable isotope labeled standards. Relative standard deviations for metabolite extraction and GC x GC-TOF-MS analysis of those analytes ranged from 13.2 to 26.3% for the universal m/z 73 trace and 7.4 to 24.5% for the analyte specific fragment ion trace.
authors.affiliation: Institute of Functional Genomics, University of Regensburg, Josef-Engert-Strasse 9, 93053 Regensburg, Germany.
authors.forename: Martin F
authors.initials: MF
authors.lastname: Almstetter
authors.forename: Inka J
authors.initials: IJ
authors.lastname: Appel
authors.forename: Michael A
authors.initials: MA
authors.lastname: Gruber
authors.forename: Claudio
authors.initials: C
authors.lastname: Lottaz
authors.forename: Birgit
authors.initials: B
authors.lastname: Timischl
authors.forename: Rainer
authors.initials: R
authors.lastname: Spang
authors.forename: Katja
authors.initials: K
authors.lastname: Dettmer
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Oefner
mesh_terms: D002849:Chromatography, Gas; D001330:Electronic Data Processing; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D016014:Linear Models; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods*; D009154:Mutation; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism; D025341:Principal Component Analysis; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D009249:NADP; D009250:NADP Transhydrogenases