Labelling analysis for ¹³C MFA using NMR spectroscopy

NMR spectroscopy is an efficient method for analyzing ¹³C labelling of cellular metabolites. The strength of it is especially the ability to provide direct quantitative positional information on the ¹³C labelling status of carbon atoms in metabolites. NMR spectroscopic methods allow also for detection of contiguously ¹³C-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 the ¹³C labelling of the biomass amino acids for metabolic flux analysis, sample preparation for NMR spectroscopy, acquiring and processing the NMR spectra, and extracting the ¹³C labelling information from the NMR data. Different NMR methods are applied depending on the ¹³C labelling strategy chosen. These strategies include uniform ¹³C labelling, positional ¹³C labelling, or a combination of both. Not only the preparation of sample for analysis of ¹³C labelling in proteinogenic amino acids in biomass is described, but also the necessary modifications to the method when analysis of ¹³C labelling in free metabolic intermediates is of interest. Finally the strategies for using the different NMR-detected ¹³C labelling data in ¹³C MFA are discussed.