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

Creek DJ, Chokkathukalam A, Jankevics A, Burgess KE, Breitling R, Barrett MP - Anal. Chem. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.

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.

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KEGG global pathway map of identified and putatively annotatedmetabolites labeled by 13C-glucose (red) and unlabeledputative metabolites arising from alternative sources (gray). Inferredpotentially active biosynthesis pathways are highlighted; black routeshave not previously been known to be active in Trypanosomabrucei. Map generated using IPath tools.30
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fig2: KEGG global pathway map of identified and putatively annotatedmetabolites labeled by 13C-glucose (red) and unlabeledputative metabolites arising from alternative sources (gray). Inferredpotentially active biosynthesis pathways are highlighted; black routeshave not previously been known to be active in Trypanosomabrucei. Map generated using IPath tools.30

Mentions: The extent and pattern of labeling in each (putative) metabolitewas determined by an automated search of all potential isotopomersaccording to accurate mass and retention time. Comparison of isotopedistributions with unlabeled samples, and with theoretical naturalisotope abundances, confirmed the presence of stable isotope labelingfor 187 metabolites (Figure 1a). Visualizationof labeled metabolites in the global metabolic network30 reveals incorporation of glucose-derived carbon into diverseareas of metabolism (Figure 2).


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

Creek DJ, Chokkathukalam A, Jankevics A, Burgess KE, Breitling R, Barrett MP - Anal. Chem. (2012)

KEGG global pathway map of identified and putatively annotatedmetabolites labeled by 13C-glucose (red) and unlabeledputative metabolites arising from alternative sources (gray). Inferredpotentially active biosynthesis pathways are highlighted; black routeshave not previously been known to be active in Trypanosomabrucei. Map generated using IPath tools.30
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472505&req=5

fig2: KEGG global pathway map of identified and putatively annotatedmetabolites labeled by 13C-glucose (red) and unlabeledputative metabolites arising from alternative sources (gray). Inferredpotentially active biosynthesis pathways are highlighted; black routeshave not previously been known to be active in Trypanosomabrucei. Map generated using IPath tools.30
Mentions: The extent and pattern of labeling in each (putative) metabolitewas determined by an automated search of all potential isotopomersaccording to accurate mass and retention time. Comparison of isotopedistributions with unlabeled samples, and with theoretical naturalisotope abundances, confirmed the presence of stable isotope labelingfor 187 metabolites (Figure 1a). Visualizationof labeled metabolites in the global metabolic network30 reveals incorporation of glucose-derived carbon into diverseareas of metabolism (Figure 2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TA, UK.

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.

Show MeSH