13C-labeled glutamine tracing and mass spectrometry analysis

Cells were seeded into 6-well plates in 2 ml of culture medium for overnight (500.000 cells per well). The next day cells were washed three times with PBS and cultured with DMEM (DU145 cells) or RPMI (LNCaP cells) medium supplemented with 2 mM [U-¹³C₅] L-glutamine (Sigma) for 10, 30 and 90 min. Three independent experiments were performed with one replicate. Targeted analysis of metabolites was performed by mass spectrometry using an ABSCIEX 5500 QTRAP equipped with SelexION for differential mobility separation (DMS) and acquired using multiple reaction monitoring (MRM) in negative mode. Cells were quenched with an ice-cold solution containing 20% methanol, 0.1% formic acid, 10 µM D4-taurine, 3 mM NaF, 1 mM phenylalanine and 100 µM EDTA. Cell extracts were then collected, frozen immediately, lyophilized overnight and resuspended in water before mass spectrometry analysis. Samples were injected onto a Hypercarb column (3 μm particle size, 4.6 x 100 mm, Thermo Fisher Scientific) at a flow rate of 0.6 mL / min on an ACQUITY UPLC System. Chromatographic separation was achieved with a gradient of ionic strength: mobile phase A contained 15 mM ammonium formate and 20 μL EDTA; mobile phase B contained 15 mM ammonium formate and a acetonitrile: isopropyl alcohol mixture (2:1). The gradient had a duration of 6 min with the following profile: 0 min, 0% B; 1 min, 0% B; 1.5 min, 20% B; 2 min, 20% B; 2.5 min, 0% B; 6 min, 0% B. Samples were ionized by electrospray into the mass spectrometer with the following source parameters: CUR: 20, CAD: 7, IS: -4500, TEM: 550, GS1: 70 and GS2: 60. DMS was used as an additional separation axis (DT: low, MD: 2-propanol, MDC: high, DMO: 3 and DR: off). DMS-related Separation Voltage (SV) and Compensation Voltage (CoV) for each metabolite was optimized before each experiment. Peak identification and chromatographic separation of all metabolites was confirmed with known standards. Peaks were integrated with ElMaven v0.6.1 (Elucidata). The ensuing peak areas were used for the calculation of metabolite concentrations and ¹³C-enrichments as previously described ⁸¹.