title: COMPLETE-MFA: complementary parallel labeling experiments technique for metabolic flux analysis.
abstract: We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-(13)C], [2-(13)C], [3-(13)C], [4-(13)C], [5-(13)C], and [6-(13)C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography-mass spectrometry (GC-MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution (13)C-flux analysis.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Metabolic Engineering and Systems Biology Laboratory, University of Delaware, 150 Academy St., Newark, DE 19716, USA.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Leighty
authors.forename: Maciek R
authors.initials: MR
authors.lastname: Antoniewicz
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D048168:Escherichia coli K12 / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D055442:Metabolome / Q000502:physiology*; D008954:Models, Biological
chemical_list: D002247:Carbon Isotopes; D005947:Glucose
keywords: (13)C tracers; Combined flux analysis; E. coli; Metabolic network; Parallel experiments