Abstract
BackgroundStable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. Measuring isotopic enrichment in metabolites provides quantitative insights into the biosynthetic network and enables flux analysis as a function of external perturbations. NMR and mass spectrometry are the techniques of choice for global profiling of stable isotope labeling patterns in cellular metabolites. However, meaningful biochemical interpretation of the labeling data requires both quantitative analysis and complex modeling. Here, we demonstrate a novel approach that involved acquiring and modeling the timecourses of 13C isotopologue data for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) synthesized from [U-13C]-glucose in human prostate cancer LnCaP-LN3 cells. UDP-GlcNAc is an activated building block for protein glycosylation, which is an important regulatory mechanism in the development of many prominent human diseases including cancer and diabetes.ResultsWe utilized a stable isotope resolved metabolomics (SIRM) approach to determine the timecourse of 13C incorporation from [U-13C]-glucose into UDP-GlcNAc in LnCaP-LN3 cells. 13C Positional isotopomers and isotopologues of UDP-GlcNAc were determined by high resolution NMR and Fourier transform-ion cyclotron resonance-mass spectrometry. A novel simulated annealing/genetic algorithm, called 'Genetic Algorithm for Isotopologues in Metabolic Systems' (GAIMS) was developed to find the optimal solutions to a set of simultaneous equations that represent the isotopologue compositions, which is a mixture of isotopomer species. The best model was selected based on information theory. The output comprises the timecourse of the individual labeled species, which was deconvoluted into labeled metabolic units, namely glucose, ribose, acetyl and uracil. The performance of the algorithm was demonstrated by validating the computed fractional 13C enrichment in these subunits against experimental data. The reproducibility and robustness of the deconvolution were verified by replicate experiments, extensive statistical analyses, and cross-validation against NMR data.ConclusionsThis computational approach revealed the relative fluxes through the different biosynthetic pathways of UDP-GlcNAc, which comprises simultaneous sequential and parallel reactions, providing new insight into the regulation of UDP-GlcNAc levels and O-linked protein glycosylation. This is the first such analysis of UDP-GlcNAc dynamics, and the approach is generally applicable to other complex metabolites comprising distinct metabolic subunits, where sufficient numbers of isotopologues can be unambiguously resolved and accurately measured.
Highlights
Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways
Metabolism of LN3 cells As with many other cancer cells in culture, 13C glucose is a major source of carbon for nucleotide riboses, amino acids such as Ala, Glu and Asp via glycolysis and the Krebs cycle, and pyrimidine rings in LnCaP-LN3 cells
The unlabeled and labeled metabolite profiles of the LN3 cells and media grown in the presence of [U-13C]-glucose were determined by NMR and mass spectrometry as described in the Methods section
Summary
Stable isotope tracing is a powerful technique for following the fate of individual atoms through metabolic pathways. We have been developing stable isotope-resolved metabolomic analysis (SIRM) for polar and non-polar metabolites in cell and tissue systems to obtain a comprehensive view of the flow of carbon or nitrogen through different metabolic pathways [5,6,7,8,9,10,11,12] This approach involves the combined use of NMR and mass spectrometry (MS), both at very high resolution, which respectively provide direct information on positional isotopomers and isotopologues (sometimes termed ‘mass isotopomers’) of labeled metabolites in an unfractionated mixture, thereby minimizing errors from sample processing [10]. The technique can be applied to any complex metabolite where a sufficient number of mass isotopologues can be identified and accurately quantified
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