NMR method for measuring carbon-13 isotopic enrichment of metabolites in complex solutions.
Isotope-based methods are commonly used for metabolic flux analysis and metabolite quantification in biological extracts. Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for these studies because NMR can unambiguously identify compounds and accurately measure (13)C enrichment. We have developed a new pulse sequence, isotope-edited total correlation spectroscopy (ITOCSY), that filters two-dimensional (1)H-(1)H NMR spectra from (12)C- and (13)C-containing molecules into separate, quantitatively equivalent spectra. The ITOCSY spectra of labeled and unlabeled molecules are directly comparable and can be assigned using existing bioinformatics tools. In this study, we evaluate ITOCSY using synthetic mixtures of standards and extracts from Escherichia coli . We show that ITOCSY has low technical error (6.6% for metabolites ranging from 0.34 to 6.2 mM) and can detect molecules at concentrations less than 10 muM. We propose ITOCSY as a practical NMR strategy for metabolic flux analysis, isotope dilution experiments, and other methods that rely on carbon-13 labeling.
Lewis
Ian A
IA
National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Karsten
Ryan H
RH
Norton
Mark E
ME
Tonelli
Marco
M
Westler
William M
WM
Markley
John L
JL
D002247:Carbon Isotopes; D002457:Cell Extracts / Q000032:analysis; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D015203:Reproducibility of Results; D012996:Solutions
D002247:Carbon Isotopes; D002457:Cell Extracts; D012996:Solutions
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