Development of a quantitative, validated capillary electrophoresis-time of flight-mass spectrometry method with integrated high-confidence analyte identification for metabolomics.
A CE-MS method was developed and validated for the quantitative analysis of negatively charged metabolites by making use of the high mass accuracy and the quantitation capabilities of a TOF mass analyzer in combination with automated feature extraction and database search. Metabolites of the central carbon metabolism were quantified with an LOD and lower LOQ (LLOQ) of 0.2-2 and 1-4 microM, respectively. The method was used to elucidate metabolic changes in the Escherichia coli deletion mutant PntAB-UdhA that lacks nicotinamide nucleotide transhydrogenase function, under both stationary and exponential growth conditions. The reproducibility of metabolite extraction and CE-TOF-MS analysis ranged from 3.7 to 22.7 and 7.9 to 22.6%, respectively, while the biological variance was 3.4-31.3%. We observed significant differences in metabolite abundance, particularly in the citrate cycle, between wild-type and mutant E. coli. Overall, more than 600 features were found by automated feature detection, which resulted in approximately 150 high-confidence metabolite identifications. Concomitant analyses with two different GC-MS methods allowed not only crossvalidation of the quantitative results obtained by the various methods, but also led to a more comprehensive coverage of the E. coli metabolome.
Timischl
Birgit
B
Institute of Functional Genomics, University of Regensburg, Regensburg, Germany.
Dettmer
Katja
K
Kaspar
Hannelore
H
Thieme
Marian
M
Oefner
Peter J
PJ
D000596:Amino Acids / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000706:statistics & numerical data; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods / Q000706:statistics & numerical data; D005798:Genes, Bacterial; D008660:Metabolism; D009154:Mutation; D009250:NADP Transhydrogenases / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000706:statistics & numerical data
D000596:Amino Acids; D009250:NADP Transhydrogenases