146 CHAPTER 5 SUPPLEMENTARY METHODS PATIENT SAMPLES This study received approval from the Amsterdam UMC Biobank under study number BUP201912. Regular diagnostic procedures were not hindered by the collection of the study biopsies. CELL LINES K562 chronic myeloid leukemia (CML) cells were obtained from the ATCC and cultured in DMEM medium supplemented with 10% FBS (Biowest, France). Cells were maintained at 37 °C and expanded in a T175 culture flaks. Nine aliquots of 10 ml cell suspension (exponential growth phase) were transferred into a 50 ml tube and centrifuged for 2 minutes at 300g and the supernatant was removed. Cells were washed twice in phosphate-buffered saline (PBS) and centrifuged for 2 minutes at 300g before being resuspended in 1 ml PBS and transferred in the aluminum vial. The vial was placed into a 50 ml tube and centrifuged for 1 minute at 300g rpm, after which PBS was removed and the pellet of cells remained on the bottom of the vial. The vials subsequently entered their respective freezing procedures, see below, and were stored at -80 °C until further processing. Cells from the colorectal cancer cell line HCT116 were cultured in biological triplicates in DMEM medium (Lonza Biowhittaker, Verviers, Belgium) containing 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml sodium penicillin and 100 µg/ml streptomycin. Cells were lysed in lysis buffer containing 9 M urea, 20 mM HEPES pH 8.0, 1 mM Na3VO4 (orthovanadate), 2.4 mM Na4P2O7 (pyrophosphate), and 1 mM Na2C3H7PO6 (β-glycerophosphate) by scraping and subsequent sonication. After lysis, protein concentration was determined using the BCA method (ThermoPierce, Rockford, IL). Cell lysate was reduced in 4 mM dithiotreitol (DTT) for 20 minutes at 60 °C, cooled to room temperature and alkylated in 10 mM iodoacetamide for 15 minutes in the dark. Next, the cell lysate was diluted to 2 M urea using 20 mM HEPES buffer pH 8.0 and digested overnight with trypsin (10 µg/mg protein) at 37 °C. Digestion was stopped in 0.1% trifluoroacetic acid (TFA). PHOSPHOPEPTIDE ENRICHMENT AND LC-MS/MS MEASUREMENT FOR PHOSPHOPROTEOMICS HCT116 cell lysate aliquots of 300-400 µg protein, K562 cell lysate aliquots of 500 µg protein and tissue lysates were reduced, alkylated and digested as described previously1. Desalted peptides were enriched for phosphopeptides using titanium oxide (TiOx) beads based using aliphatic hydroxyl-acid modified metal oxide chromatography2,3. In brief, 500 µg desalted peptides (1 µg/µl in 80% ACN, 0.1% TFA) were mixed with 500 µl washing buffer (80% ACN, 0.1% TFA containing 300 mg/ml lactic acid) and applied to 2.5 mg TiOx beads (GL sciences, 10 µm) packed in a 200 µl STAGE tip containing a 16G empore C8 membrane plug (3M, St Paul, MN). The STAGE tip was washed with 200 µl washing buffer followed by 200 µl 80% ACN, 0.1% TFA. Phosphopeptides were eluted in two steps in 50 µl 0.5% and 5% piperidine (Fisher Scientific) and were quenched in 100 µl 20% H3PO4. All steps were performed by centrifugation (1500 x
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