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Development of a quantitative, validated Capillary electrophoresis‐time of flight – mass spectrometry method with integrated high‐confidence analyte identification for metabolomics
Author(s) -
Timischl Birgit,
Dettmer Katja,
Kaspar Hannelore,
Thieme Marian,
Oefner Peter J.
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700517
Subject(s) - metabolomics , metabolite , metabolome , chromatography , analyte , chemistry , capillary electrophoresis , mass spectrometry , biochemistry
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 µM, 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 ∼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.