Metabolic Labeling of Ovarian Cancer Cells

Serous ovarian cancer cell line SKOV3 and its high metastatic derivative SKOV3-ip were obtained from the American Type Culture Collection (ATCC). These two ovarian cancer cell lines were grown in RPMI1640 cell media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen). 293T cell line was obtained from the Institute of Cell Biology Academic Sinica (Shanghai, China). 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. All cells were cultured with 1% penicillin/streptomycin at 37°C in a humidified 5% CO₂ incubator. For stable isotope labeling experiments, cells were cultured for at least six doublings in the RPMI1640 medium with 10% fetal bovine serum, containing 20 mM L-glutamine (either ¹⁴N or amide-¹⁵N-Gln) to achieve complete incorporation of labeled N-glycans [41] (link). Amide-¹⁵N-Gln (98% purity) was purchased from Cambridge Isotopes, Inc. (Andover, MA, USA).