Gel-based experiment (human)

Frozen cell pellets from 4x15 cm cell culture plates were lysed in Glycerol buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 5 mM EDTA, 0.5 % Triton-X-100, 10 % Glycerol, 1x protease inhibitor, 1x phosphatase inhibitor) for 30 min at 4° C with end-over-end rotation. Lysates were cleared from cell debris by centrifugation prior to adjustment of protein concentrations between the samples and overnight immunoprecipitation at 4° C with pre-equilibrated anti-HA-agarose (Sigma). Agarose beads were washed five times with Glycerol buffer followed by elution of proteins with 3x loading buffer and boiling of the samples at 95° C. Samples were then analyzed by SDS-PAGE (BioRad’s 4-20 % gels) followed by in-gel tryptic digestion. SDS-PAGE gel lines were cut in 12 equal size bands, further chopped in smaller pieces and placed in 96 well plates (one band per well). Gel pieces were washed with 50 mM ammonium bicarbonate (ABC)/50 % EtOH buffer followed by dehydration with EtOH, reduction of proteins with 10 mM DTT in 50 mM ABC at 56° C for 1 hr and alkylation of proteins with 55 mM iodacetamide in 50 mM ABC at room temperature for 45 min. Prior to overnight trypsin-digest (12 ng/ul trypsin in 50 mM ABC, Promega) at 37° C, gel pieces were washed and dehydrated as before. Peptide were extracted from gel pieces with 30 % acetonitrile/3 % trifluoroacetic acid (TFA), 70 % acetonitrile and finally 100 % acetonitrile followed by desalting on custom-made C18-stage tips. Using an Easy-nLC1200 liquid chromatography (Thermo Scientific), peptides were loaded onto 75 µm x 15 cm fused silica capillaries (New Objective) packed with C18AQ resin (Reprosil- Pur 120, 1.9 µm, Dr. Maisch HPLC). Peptide mixtures were separated using a gradient of 5%–33% acetonitrile in 0.1% acetic acid over 75 min and detected on an Q Exactive HF mass spectrometer (Thermo Scientific). Dynamic exclusion was enabled for 30 s and singly charged species or species for which a charge could not be assigned were rejected.