Labelling analysis for 13C MFA using NMR spectroscopy

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Abstract

NMR spectroscopy is an efficient method for analyzing13C labelling of cellular metabolites. The strength of it is especially the ability to provide direct quantitative positional information on the13C labelling status of carbon atoms in metabolites. NMR spectroscopic methods allow also for detection of contiguously13C-labelled fragments in the carbon backbones of the metabolites. Furthermore, the recent developments of NMR spectroscopy hardware have substantially improved the sensitivity of the methods. In this chapter we describe a method for analyzing the13C labelling of the biomass amino acids for metabolic flux analysis, sample preparation for NMR spectroscopy, acquiring and processing the NMR spectra, and extracting the13C labelling information from the NMR data. Different NMR methods are applied depending on the13C labelling strategy chosen. These strategies include uniform13C labelling, positional13C labelling, or a combination of both. Not only the preparation of sample for analysis of13C labelling in proteinogenic amino acids in biomass is described, but also the necessary modifications to the method when analysis of13C labelling in free metabolic intermediates is of interest. Finally the strategies for using the different NMR-detected13C labelling data in13C MFA are discussed.

Original languageEnglish
Title of host publicationMetabolic Flux Analysis
EditorsJ. Krömer, L. Nielsen, L. Blank
PublisherSpringer
Pages143-164
Number of pages22
ISBN (Electronic)978-1-4939-1170-7
ISBN (Print)978-1-4939-1169-1
DOIs
Publication statusPublished - 12 Jul 2014
MoE publication typeA3 Part of a book or another research book

Publication series

SeriesMethods in Molecular Biology
Volume1191
ISSN1064-3745

Keywords

  • C
  • C, H] COSY
  • HSQC
  • Metabolic flux
  • MFA
  • NMR
  • ZQF-TOCSY

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