title: Implementation of data-dependent isotopologue fragmentation in13C-based metabolic flux analysis.
abstract: A novel analytical approach based on liquid chromatography coupled to quadrupole time of flight mass spectrometry, employing data-dependent triggering for analysis of isotopologue and tandem mass isotopomer fractions of metabolites of the primary carbon metabolism was developed. The implemented QTOFMS method employs automated MS/MS triggering of higher abundant, biologically relevant isotopologues for generating positional information of the respective metabolite. Using this advanced isotopologue selective fragmentation approach enables the generation of significant tandem mass isotopomer data within a short cycle time without compromising sensitivity. Due to a lack of suitable reference material certified for isotopologue ratios, a Pichia pastoris cell extract with a defined13C distribution as well as a cell extract from a13C-based metabolic flux experiment were employed for proof of concept. Moreover, a method inter-comparison with an already established GC-CI-(Q)TOFMS approach was conducted. Both methods showed good agreement on isotopologue and tandem mass isotopomer distributions for the two different cell extracts. Graphical abstract Schematic overview of data-dependent isotopologue fragmentation for acquisition of isotopologue and tandem mass isotopomer fractions.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences - BOKU Vienna, Muthgasse 18, 1190, Vienna, Austria.|Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Teresa
authors.initials: T
authors.lastname: Mairinger
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences - BOKU Vienna, Muthgasse 18, 1190, Vienna, Austria. stephan.hann@boku.ac.at.|Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190, Vienna, Austria. stephan.hann@boku.ac.at.
authors.forename: Stephan
authors.initials: S
authors.lastname: Hann
mesh_terms: D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D007553:Isotope Labeling / Q000379:methods; D064688:Metabolic Flux Analysis / Q000379:methods*; D010843:Pichia / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D057188:Workflow
chemical_list: D002247:Carbon Isotopes
keywords: 13C-based metabolic flux analysis; Data-dependent fragmentation; Isotopologue ratio