Quantifying Cellular Metabolism via LC-MS

For cell-based ¹³C₆ metabolite labeling analysis, around 90% confluent H1299 cells were washed by glucose-free culture medium (Gibco, 11966-025) and cultured in medium containing ¹³C₆-glucose (10 mM) (Cambridge Isotope Laboratories, CLM-1396) for 30 min. High-resolution LC–MS/MS was used for analyzing the levels of ¹³C₆-labeled intracellular metabolites^{11 (link)}.
For label-free xenograft metabolite analysis, the xenograft tissue samples were homogenized in 150 μl of extraction solution (half methanol and half water) with 250 ng ml⁻¹ 4-nitrobenzoic acid and debrisoquine as internal standard for negative and positive modes, respectively, followed by adding 150 μl of acetonitrile. After incubation for 30 min at −20 °C, the samples were centrifuged at a speed of 16,000g for 30 min at 4 °C. The standards—namely inosine 5′-monophosphate disodium salt (IMP, I4625), adenosine 5′-monophosphate disodium salt (AMP, 01930), guanosine 5′-monophosphate disodium salt (GMP, G8377), uridine 5′-monophosphate disodium salt (UMP, U6375) and cytidine 5′-monophosphate disodium salt (CMP, C1131)—were obtained from Sigma-Aldrich. The samples were transferred to MS vials for LC–MS analysis by QTRAP 7500 System (SCIEX). Data were acquired and normalized to the internal standards and then analyzed with SCIEX OS software.