Shotgun proteomics

HeLa cells stably expressing CNN2-APEX2 were grown at 37°C in DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate. Cells were differentially treated with 5 µM NMS-873 for 15 min followed by 1 h 1 mM LLOMe (Sigma) and 2 h washout wihout any drugs. Proximity labeling was performed essentially as described before (Korver et al., 2019). Briefly, cells were incubated with 500 µM Biotin-Phenol during the last 30 min and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop the biotinylation reaction, they were washed 3x with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in DPBS) and 3x with PBS. All further steps were performed at 4°C unless indicated otherwise. After cell harvest with 0.25% Trypsin/EDTA (Thermo Fisher Scientific), cells were counted and heavy- and light-labelled cells were mixed at a 1:1 ratio based on total cell numbers. After centrifugation, the resulting cell pellets were lysed in APEX-RIPA (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate supplemented with 10 mM sodium ascorbate, 1 mM sodium azide, 1 mM Trolox and protease inhibitors (Roche Complete)). Samples were sonicated 2x for 1 s, spun down at 10,000xg for 10 min before application to streptavidin agarose resin (Thermo Fisher Scientific) and incubation with overhead shaking overnight. Subsequently, samples were washed 3x in APEX-RIPA buffer and 3x in 3 M Urea buffer (in 50 mM ABC) followed by incubation with TCEP (5 mM final) for 30 min at 55°C with shaking. After alkylation with IAA (10 mM final) for 20 min at room temperature in the dark the reaction was quenched with DTT (20 mM final). Samples were washed 2x with 2 M Urea (in 50 mM ABC) before trypsin digestion overnight at 37°C (20 µg/ml final). The resin was spun down and supernatants containing digested peptides were collected. After washing the resin 2x with 2 M Urea and pooling all supernatants the samples were acidified with TFA (1% final).