Shotgun proteomics

BV2 cells were cultured in full medium in 10 cm cell culture dishes until they were confluent. MLN4924 (500 nM), CSN5i-3 (1 μM), or solvent control (0.01% DMSO) were added and the culturing continued for 6 h. After the incubation, cells were washed once with PBS, removed from the cell culture plate with a scraper, collected in tubes, and centrifuged in 1.5 ml Eppendorf tubes for 3 min to remove remaining PBS buffer, snap-frozen, and stored at -80 °C until further processing. Thereafter cells were collected and lysed in urea buffer (9 M Urea, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1x Roche protease inhibitor cocktail, product # 11836153001) followed by short sonification. Samples were cleared by centrifugation and protein amounts were adapted. Protein reduction was performed with dithiothreitol (DTT; 5 mM final) for 25 min at 56°C and protein alkylation by the addition of iodoacetamide (14 mM final) for 30 min at room temperature. Protein mixtures were quenched with DTT and diluted 1:5 with 1 M Tris-HCl, pH 8.2. For increased peptide recovery, proteins were digested at room temperature for 3 h with LysC (FUJIFILM, 2 µL/100 µg protein) before overnight tryptic digest at 37°C (0.5 µg/100µg protein). The following day, digestion was stopped with 10% trifluoroacetate (TFA). To increase analysis depth, samples were pre-fractioned by a C18-SCX custom-made stage tip. Fractions were eluted stepwise with increasing NH4AcO concentrations (20 mM to 500 mM) and desalted on a separate C18 stage tip. Desalted peptides were loaded on a custom-made 75 mm x 15 cm fused silica capillary filled with C18-AQ resin (Reprosil Pur 120 HPLC column, 1.9 µm, Dr. Maisch HPLC GmbH, Ammerbuch, Germany) using an Easy-nLC1200 liquid chromatography. Samples were separated for 140 min with a 2.4-80% acetonitrile gradient in 0.1 % formic acid using a Q Exactive HF mass spectrometer (MS; ThermoFisher Scientific).