Stable isotope-assisted metabolomics for network-wide metabolic pathway elucidation

Abstract

The combination of high-resolution LC-MS-based untargeted metabolomics with stable isotope tracing provides a global overview of the cellular fate of precursor metabolites. This methodology enables detection of putative metabolites from biological samples and simultaneous quantification of the pattern and extent of isotope labeling. Labeling of Trypanosoma brucei cell cultures with 50% uniformly (13)C-labeled glucose demonstrated incorporation of glucose-derived carbon into 187 of 588 putatively identified metabolites in diverse pathways including carbohydrate, nucleotide, lipid, and amino acid metabolism. Labeling patterns confirmed the metabolic pathways responsible for the biosynthesis of many detected metabolites, and labeling was detected in unexpected metabolites, including two higher sugar phosphates annotated as octulose phosphate and nonulose phosphate. This untargeted approach to stable isotope tracing facilitates the biochemical analysis of known pathways and yields rapid identification of previously unexplored areas of metabolism.

Figure 1

(a) Schematic representation of the method. Red dots represent labeled metabolites. (b) Labeling pattern in succinate, aspartate, and orotate biosynthesis. In contrast to the expected predominant 2- and 4-carbon labeling from the TCA cycle, the majority of molecules contain three labeled carbons, consistent with a fermentative source of succinate. (c) Heavy-isotope labeling patterns in the new trypanosomal metabolites putatively annotated as octulose phosphate and nonulose phosphate. C represents the unlabeled control, and L represents the ¹³C-glucose labeled sample.

Figure 2

KEGG global pathway map of identified and putatively annotated metabolites labeled by ¹³C-glucose (red) and unlabeled putative metabolites arising from alternative sources (gray). Inferred potentially active biosynthesis pathways are highlighted; black routes have not previously been known to be active in Trypanosoma brucei. Map generated using IPath tools.