Isotope tracing assisted metabolic profiling: Application to understanding HSP60 silencing mediated tumor progression

Isotope-tracing facilitates the understanding of metabolic regulation in biological systems. Depending on the selection of tracers, some essential metabolites cannot be traced. A comprehensive understanding of the regulated pathways can only be achieved with focus beyond labeled metabolites. The isotope tracing assisted metabolic profiling described here is a platform for high throughput mapping of isotope labeled metabolites with simultaneous metabolomics profiling. This approach incorporates an in-house MS/MS library for metabolite identification and ID-based quantitation. An "Isotopic" software was developed to generate potential labeled isotopomers. Using this platform, a total of 394 metabolites were reliably identified based on MS/MS confirmation in 3 million 293 T cells, among which 54 and 43 metabolites were discovered to carry extensive labels (>2%) from ¹³C₆-glucose and ¹³C₅-glutamine respectively. Citrate flowing into malate shuttle was also observed. More interestingly, the rate-limiting step in NAD and UDP-GlcNAc biosynthesis was clearly observed according to time course labeling. In HSP60 knockdown cell lines, enhanced purine and pyrimidine biosynthesis were confirmed by the abundance and labeling percentages of intermediate metabolites.