Kinetic isotope tracing of de novo proteogenic amino acids and glycerol in A549 cells using [13C6]glucose

In this experiment, we aimed to monitor the metabolic intermediates including de novo amino acids in A549 cells upon exposure to U-C13 glucose. We observed that A549 cells produce kinetically labelled lactate, glycerol, and several amino acids that were incorporated in protein synthesis and contributed to biomass production. A549 cells grown on U-C13 glucose were used to extract the culture filtrate, methanolic metabolite fractions, and acid hydrolysate of proteins. These extracted metabolites were derivatised and analysed using gas chromatography and mass spectrometer (GC-MS). The GC-MS data were baseline corrected using MetAlign and the accurate mass isotopomer distribution (MIDs) were extracted for all labelled analytes. The mass isotopomer abundances were measured using Isocor software (Millard et al., 2012).