L glutamine 13c5

¹³C enrichment in intracellular metabolites was measured upon cell incubation with uniformly-labeled glucose (D-Glucose-¹³C₆, Sigma-Aldrich 389374) or glutamine (l-Glutamine-¹³C₅, Sigma-Aldrich 605166). Briefly, cells were seeded on poly-D-lysine–coated glass coverslips in complete DMEM-F12 medium (34 mmol/L glucose, 2 mmol/L glutamine). A total of 24 hours later, cells were thoroughly washed and placed in glucose-free and glutamine-free DMEM-F12 medium (BioWest L0091) supplemented with either 34 mmol/L labeled glucose and 2 mmol/L unlabeled glutamine or 2 mmol/L labeled glutamine and 34 mmol/L unlabeled glucose, for 24 and 48 hours. A control coverslip without cells was added for each experiment. Metabolites were extracted at −20°C in acetonitrile/methanol/water (2:2:1) containing formic acid (0.1%) and further processed (see Supplementary Materials and Methods).

SU. 8686 pancreatic cancer cells were cultured for 12 h or 24 h in L-glutamine-¹³C₅ (2 mM, Cat. #605166, Sigma‒Aldrich) or L-glutamine-¹⁵N₂ (Sigma‒Aldrich, 490032). Cells cultured in unlabeled glutamine (Sigma‒Aldrich, Cat.# G3126) were used as controls. A total of 1 × 10⁷ cells were sent for analysis. Samples were prepared as previously described [35 (link)]. LC‒MS/MS analyses were performed using a UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to a Q Exactive mass spectrometer (Thermo). Mobile phase A was 0.1% formic acid in water for positive mode and 5 mmol/L ammonium acetate in water for negative mode, and mobile phase B was acetonitrile. The elution gradient was set as follows: 0–1.0 min, 1% B; 1.0–8.0 min, 1–99% B; 8.0–10.0 min, 99% B; 10.0–10.1 min, 99–1% B; 10.1–12 min, 1% B. The flow rate was 0.5 mL/min. The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration [36 (link)]. Then, an in-house MS2 database (BiotreeDB) was applied for metabolite annotation. The cutoff for annotation was set at 0.3.

SU.8686 cells were cultured for 12 or 24 h in media containing L-glutamine-
¹⁵N
₂ (Cat. #490032; Sigma-Aldrich) or L-Glutamine-
¹³C
₅ (Cat. #605166; Sigma-Aldrich). Cells cultured in unlabelled glutamine were used as controls. A total of 1×10
⁷ cells were collected for analysis. For sample preparation, 1000 μL of extraction solution (acetonitrile:methanol:water=2:2:1) containing an isotopically labelled internal standard mixture was added to the sample. LC-MS/MS analyses were performed using a UHPLC System (1290; Agilent Technologies) with a UPLC HSS T3 column (2.1 mm× 100 mm,1.8 μm) coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific). The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program that was developed using R and based on XCMS for peak detection, extraction, alignment, and integration
[16] (link). M+ (M0, M1 to Mn) represents the number of C or N atoms in the metabolites labelled by the isotopes. The fractional abundances of the mass isotopomers defined by M0, M1, and Mn were calculated.

Cells (6 × 10⁶) were plated onto two 15-cm dishes and cultured in standard medium for 24 h. Cells were washed in PBS and the medium was then replaced by either fresh DMEM (no glucose) with 1 mM sodium pyruvate and 10 mM ¹³C₂-[1,2]-D-glucose (Sigma) or DMEM (high glucose with pyruvate, no glutamine) supplemented with 2 mM ¹³C₅-L-glutamine (Sigma) for 24 h. For aspartate labelling, cells were cultured in standard medium supplemented with 0.1 mM ¹³C₄-L-aspartate for 48 h.