Dual strategy for ¹³C-Metabolic flux analysis of central carbon and energy metabolism in Mammalian cells based on LC-isoMRM-MS

Central carbon and energy metabolism are the most concerned metabolic pathways in ¹³C-Metabolic flux analysis (¹³C-MFA). However, some α-keto acids, ribonucleoside triphosphate (NTPs) and deoxyribonucleoside triphosphate (dNTPs) involved in central carbon and energy metabolism pathways were unstable or reactive, leading to inaccurate metabolic flux analysis. To achieve accurate ¹³C-MFA of central carbon and energy metabolism, we proposed a dual strategy for the detection of 101 metabolites in glucose metabolism pathways. N-Methylphenylethylamine (MPEA) was utilized for derivatization of 4 carboxyl (α-keto acids) and 8 phosphate metabolites (NTPs and dNTPs). After derivatization, the MPEA derivatives were investigated to be stable for 4 weeks under 4 °C and detected with high intensity in ∼10⁴ cells. On the other hand, we analyzed an additional 89 metabolites in central carbon and energy metabolic pathways were directly analyzed by liquid chromatography tandem mass spectrometry (LC-MRM-MS). The limit of detection (LODs) of our method were as low as 0.05 ng/mL and the linear range was at least two orders of magnitude with determination coefficient (R²) > 0.9701. The relative standard divisions (RSDs) of intra- and inter-day of 95% metabolites were below 20%. In addition, the isotope list of 82 detected metabolites in central carbon and energy metabolism were generated according to isotopologues and isotopomers for each metabolite resulting from ¹³C incorporation. Accurate assessment of mass isotopomer distributions (MIDs) of intracellular ¹³C-labeled metabolites was achieved in [U–¹³C]-glucose cultured HepG2 cells by our dual strategy. Finally, we performed MID analysis of 101 metabolites in central carbon and energy metabolism. Overall, this dual method is reproducible and robust for application on ¹³C-MFA and has a great potential for studying clinical isotope labeled samples.