title: 13C metabolic flux analysis of microbial and mammalian systems is enhanced with GC-MS measurements of glycogen and RNA labeling.
abstract: 13C metabolic flux analysis (13C-MFA) is a widely used tool for quantitative analysis of microbial and mammalian metabolism. Until now,13C-MFA was based mainly on measurements of isotopic labeling of amino acids derived from hydrolyzed biomass proteins and isotopic labeling of extracted intracellular metabolites. Here, we demonstrate that isotopic labeling of glycogen and RNA, measured with gas chromatography-mass spectrometry (GC-MS), provides valuable additional information for13C-MFA. Specifically, we demonstrate that isotopic labeling of glucose moiety of glycogen and ribose moiety of RNA greatly enhances resolution of metabolic fluxes in the upper part of metabolism; importantly, these measurements allow precise quantification of net and exchange fluxes in the pentose phosphate pathway. To demonstrate the practical importance of these measurements for13C-MFA, we have used Escherichia coli as a model microbial system and CHO cells as a model mammalian system. Additionally, we have applied this approach to determine metabolic fluxes of glucose and xylose co-utilization in the E. coli ΔptsG mutant. The convenience of measuring glycogen and RNA, which are stable and abundant in microbial and mammalian cells, offers the following key advantages: reduced sample size, no quenching required, no extractions required, and GC-MS can be used instead of more costly LC-MS/MS techniques. Overall, the presented approach for13C-MFA will have widespread applicability in metabolic engineering and biomedical research.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Long
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Au
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA.
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Gonzalez
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, Newark, DE 19716, USA. Electronic address: mranton@udel.edu.
authors.forename: Maciek R
authors.initials: MR
authors.lastname: Antoniewicz
mesh_terms: D000818:Animals; D016466:CHO Cells; D002247:Carbon Isotopes / Q000493:pharmacokinetics; D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods*; D003412:Cricetulus; D004926:Escherichia coli / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006003:Glycogen / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D064688:Metabolic Flux Analysis / Q000379:methods*; D055442:Metabolome / Q000502:physiology; D008954:Models, Biological; D010427:Pentose Phosphate Pathway / Q000502:physiology*; D012313:RNA / Q000378:metabolism*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics
chemical_list: D002247:Carbon Isotopes; D019275:Radiopharmaceuticals; D012313:RNA; D006003:Glycogen
keywords: Flux estimation; Glucose and xylose co-utilization; Metabolism; Pentose phosphate pathway; Stable-isotope labeling