title: Gas Chromatography-Quadrupole Time-of-Flight Mass Spectrometry-Based Determination of Isotopologue and Tandem Mass Isotopomer Fractions of Primary Metabolites for (13)C-Metabolic Flux Analysis.
abstract: For the first time an analytical work flow based on accurate mass gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOFMS) with chemical ionization for analysis providing a comprehensive picture of (13)C distribution along the primary metabolism is elaborated. The method provides a powerful new toolbox for (13)C-based metabolic flux analysis, which is an emerging strategy in metabolic engineering. In this field, stable isotope tracer experiments based on, for example, (13)C are central for providing characteristic patterns of labeled metabolites, which in turn give insights into the regulation of metabolic pathway kinetics. The new method enables the analysis of isotopologue fractions of 42 free intracellular metabolites within biotechnological samples, while tandem mass isotopomer information is also accessible for a large number of analytes. Hence, the method outperforms previous approaches in terms of metabolite coverage, while also providing rich isotopomer information for a significant number of key metabolites. Moreover, the established work flow includes novel evaluation routines correcting for isotope interference of naturally distributed elements, which is crucial following derivatization of metabolites. Method validation in terms of trueness, precision, and limits of detection was performed, showing excellent analytical figures of merit with an overall maximum bias of 5.8%, very high precision for isotopologue and tandem mass isotopomer fractions representing >10% of total abundance, and absolute limits of detection in the femtomole range. The suitability of the developed method is demonstrated on a flux experiment of Pichia pastoris employing two different tracers, i.e., 1,6(13)C2-glucose and uniformly labeled (13)C-glucose.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences-BOKU Vienna , Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology (acib) , Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Teresa
authors.initials: T
authors.lastname: Mairinger
authors.affiliation: Austrian Centre of Industrial Biotechnology (acib) , Muthgasse 11, 1190 Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU Vienna , Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Steiger
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU Vienna , Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Justyna
authors.initials: J
authors.lastname: Nocon
authors.affiliation: Austrian Centre of Industrial Biotechnology (acib) , Muthgasse 11, 1190 Vienna, Austria.|Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU Vienna , Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Diethard
authors.initials: D
authors.lastname: Mattanovich
authors.affiliation: Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna , Waehringerstrasse 38, 1090 Vienna, Austria.
authors.forename: Gunda
authors.initials: G
authors.lastname: Koellensperger
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences-BOKU Vienna , Muthgasse 18, 1190 Vienna, Austria.|Austrian Centre of Industrial Biotechnology (acib) , Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
mesh_terms: D002247:Carbon Isotopes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D064688:Metabolic Flux Analysis; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D002247:Carbon Isotopes; D013482:Superoxide Dismutase