91 (Phospho)proteomics biomarkers for sunitinib response in RCC CONTROL SAMPLES As quality control samples, the colorectal cancer cell line HCT116 and a reference sample of tissue-mixture (containing pooled lysates of tumor samples of colorectal cancer, melanoma, non-small cell lung cancer and hepatocellular carcinoma) were used. HCT116 cells were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 U/ml sodium penicillin and 100 µg/ml streptomycin, and maintained at 37 °C. Plated cells were washed twice with phosphate-buffered saline (PBS) and lysed using 9M urea buffer. Cells were scraped and the lysate was sonicated and centrifuged for 15 minutes at maximum speed. Aliquots of lysate were stored at -80 °C. Further processing was done as described before. IMMUNOPRECIPITATION AND PROTEIN IDENTIFICATION Tumor samples were processed in 3 batches, each containing samples from patients with sensitive and resistant tumors. Immunoprecipitation (IP) of tyrosine phospho-peptides was performed using the PTMScan kit (P-Tyr-1000) from Cell Signaling Technology (Leiden, The Netherlands) as described elsewhere32,34. Briefly, lyophilized phosphopeptides were dissolved in IAP buffer (20 mM Tris-HCl pH 7.2, 10 mM sodium phosphate and 50 mM NaCl) and incubated with 2 μl P-Tyr-1000 beads per mg protein at 4 °C for 2 h. After washing in cold IAP buffer and Milli-Q water, peptides were eluted from the beads in two steps in 0.15% TFA, desalted in 20 μl Proxeon Stage Tips (Thermo Scientific) using 0.1% TFA, eluted with 80% ACN/0.1% TFA into LC autosampler vials, and stored at 4 °C until LC-MS/MS measurement on the same day. Peptides were separated on a pepmap Acclaim column (75 um ID x 500 mm, 1.9 um C18) connected to a pepmap Acclaim trap column (75 um ID x 10 mm 3 um C18) and running at 300 nl/min as described elsewhere32,33 on an Ultimate 3000 nanoLC- (Dionex LC-Packings, Amsterdam, The Netherlands) connected to a Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany) using a 2hr gradient (8-32% acetonitrile in 0.1% formic acid). Intact masses were measured at resolution 70,000 (at m/z 200) in the Orbitrap analyser using an AGC target value of 3E6 charges. The top 10 peptide signals (charge-states 2+ and higher) were submitted to MS/MS in the HCD (higher-energy collision) cell (1.4 u-amu isolation width, 25% normalized collision energy). MS/MS spectra were acquired at resolution 17.500 (at m/z 200) in the Orbitrap using an AGC target value of 1E6 charges, MaxIT of 80 ms and an underfill ratio of 0.1%. Dynamic exclusion was applied with a repeat count of 1 and an exclusion time of 30 s. LC-MS/MS spectra were searched against the Uniprot human reference proteome FASTA file (release August 2015, 62447 entries, no fragments) using MaxQuant 1.5.2.835. Enzyme specificity was set to trypsin and up to two missed cleavages were allowed. Cysteine carboxamidomethylation (Cys, +57.021464 Da) was treated as fixed modification and serine, threonine and tyrosine phosphorylation (+79.966330 Da), methionine oxidation (Met, +15.994915 Da) and N-terminal acetylation (N-terminal, +42.010565 Da) as variable modifications. Peptide precursor ions were searched with a maximum mass deviation of 4.5 ppm and fragment ions with a maximum mass deviation of 20 ppm. Peptide, protein and site identifications were filtered at a false discovery rate (FDR) of 1% using the decoy database strategy. The minimal 4
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