Analysis away from metabolic genetics from inside the medical breast tumor proteomes

Analysis away from metabolic genetics from inside the medical breast tumor proteomes

Cells were grown in glutamine and glucose-free RPMI, supplemented either with dos mmol/L 13 C5 or 15 N2-glutamine or 5 mmol/L 13 Csix– d -glucose (Cambridge Isotope Laboratories), 10% dialyzed FBS, and 1% antibiotics (penicillin–streptomycin). After 24-hour growth, metabolites were extracted with methanol: acetonitrile: water (5:3:2) and the lysates were rotated at 4°C for 10 minutes and centrifuged at 14,000 ? g for 10 minutes at 4°C. For exometabolome, the cells were cultured in with 2 mmol/L glutamine and 25 mmol/L glucose for 72 hours. Exometabolome was extracted with acetonitrile: water (4:1), vortexed vigorously, and centrifuged at 14,000 ? g for 10 minutes at 4°C. LC separation of metabolites was performed using SeQuant ZIC-pHILIC (150 ? 2.1 mm, 5 ?mol/L) connected to a SeQuant ZIC-pHILIC guard column (20 ? 2.1 mm, 5 ?mol/L; Merck) with a flow rate 0.1 mL/minute on the Ultimate 3000 UHPLC (Dionex, Thermo Scientific), with buffer A (95% acetonitrile) and buffer B (50 mmol/L ammonium carbonate, pH 10, 5% acetonitrile). […]