3.4. Quenching and Extraction

Simultaneous metabolism quenching and metabolite extraction employing ice-cold methanol was used for targeted metabolomics studies of adherent cell lines. The whole procedure was performed on ice. In the first step, the cell culture medium was aspirated and cells were washed quickly with 0.5 mL of PBS (pH 7.4) twice to remove any remaining culture medium. Then, 0.75 mL of 100% ice-cold methanol (−80 °C, LC-MS grade) containing proline-d3 (IS, c = 25 µM) was added to each well on a plate. The plates were incubated on ice for 10 min. Subsequently, cells were harvested using a rubber-tipped cell scraper. Cell lysate/methanol mixture was transferred to an Eppendorf tube, vortexed, and centrifuged (14,000× g; 4 °C; 10 min). The supernatant was transferred to an HPLC vial and subjected to LC-MS analysis. The concentrations of target metabolites were normalized to total protein content determined by BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA). Figure 2 shows all steps of the workflow in the LC-MS-based targeted analysis of proline, glutamic acid, and arginine in mammalian cell culture.