Computational protocol: Ecto-5′-nucleotidase (CD73) is a biomarker for clear cell renal carcinoma stem-like cells

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Protocol publication

[…] Cells were cultured in SILAC® RPMI-1640 medium, either containing 10% dialyzed fetal bovine serum and supplemented with 100 μg/mL 13C615N4-L-arginine (Invitrogen), designated as ‘heavy’ medium, or containing 10% fetal bovine serum and supplemented with 100 μg/mL regular arginine (Invitrogen), designated as ‘light’ medium. Cultures were maintained for 2 weeks to achieve complete labeling of cellular proteins with ‘heavy’ or ‘light’ amino acids before sorting for Rhohigh or Rholow cells, respectively. Separate suspensions of sorted Rhohigh and Rholow cells were washed three times with ice-cold PBS and then suspended in 8 M urea containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and sonicated. After centrifugation for 30 min at 20,000 × g in a bench-top centrifuge (Thermo Fisher Scientific, Waltham, MA), the supernatants were collected and kept at −80°C for analysis. Protein concentrations were measured using the Bradford method. Extracted protein samples from heavy Rhohigh cells and light Rholow cells were then combined at a 1:1 ratio. In-solution digestion was performed as follows. Briefly, 100 μg of protein mixture was dissolved in 8 M urea with 25 mM NH4HCO3 (Sigma), reduced with 10 mM DTT for 1 h, alkylated with 40 mM iodoacetamide in the dark for 45 min at room temperature, and then the iodoacetamide was quenched with 40 mM DTT for 30 min at room temperature. After diluting the 8 M urea with 25 mM NH4HCO3 to 1.6 M, sequence grade trypsin (Promega, Madison, WI) was added at a ratio of 1:30 and the protein mixture was digested at 37°C overnight. Tryptic digestion was stopped by the addition of formic acid to a 1% final concentration. Digests were centrifuged at 16,000 × g for 10 min prior to analysis. The supernatant was analyzed by 2D-LC on an LTQ Orbitrap XL (Thermo Fisher Scientific) []. The MS/MS data were searched against a human protein database downloaded from the NCBI database using the SEQUEST program (Thermo Fisher Scientific). The annotations of proteins were obtained from the Swiss-Prot and TrEMBL protein databases. For proteins without descriptions, annotations were obtained by searching the IPI, Swiss-Prot and TrEMBL protein database with BlastP for homologous proteins with descriptions. The PANTHER classification system was used for protein sorting with slight modification where a few protein groups with similar annotation were combined. […]

Pipeline specifications

Software tools Comet, BLASTP
Databases UniProt
Application MS-based untargeted proteomics
Organisms Homo sapiens, Homo sapiens/Mus musculus xenograft, Mus musculus
Diseases Carcinoma, Renal Cell, Diabetes Mellitus, Immunologic Deficiency Syndromes, Neoplasms