Isotopic tracer, a powerful technique for metabolic pathway analysis, is currently widely applied in metabolic flux analysis. However, the qualitative and quantitative analyses of13C-labeled metabolites pose great challenges, especially in complex biological sample matrices. Here, we present an integrated method for the qualitative and quantitative analyses of various isotopologues and isotopomers of13C-labeled nonessential amino acids (NEAAs) in HepG2 cells incubated with13C5-glutamine (Gln) based on ultra-high-performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS). First, accurate mass-to-charge (m/z) values of protonated isotopologues and elution time of standards were simultaneously analyzed to characterize13C-labeled NEAAs by high-resolution Orbitrap MS in the parallel reaction monitoring (PRM) mode. Second, isotopologues and isotopomers of13C-labeled NEAAs were investigated in HepG2 cells incubated with13C5-Gln at different time points. Ultimately, a total of 66 multiple reaction monitoring (MRM) transitions were performed by UHPLC coupled with triple quadrupole MS. Among them, 29 MRM transitions were monitored for pure metabolites (unambiguously identified). The other 37 MRM transitions were monitored for mixtures with exactly identical MRM transitions and retention time. The application of targeted profiling of13C-labeled NEAAs in the dynamic13C-labeling experiment indicated that the concentration-time profiles of NEAAs were different from each other. The concentrations of most13C-labeled Gln, Glu, Pro, and Asp altered after13C5-Gln incubation, indicating that Gln plays a fundamental role in the biosynthesis of Glu, Pro, and Asp. The proposed PRM-MRM combination mode LC-MS approach is expected to provide valuable insights into analyses of isotope-labeled metabolites in isotope tracer experiments.
Scopus Subject Areas
- Analytical Chemistry