Shotgun proteomics

Proximity labeling: Cells were supplemented with 500 µM biotin-phenol (IrisBiotech) for 30 min at 37°C before addition of 1 mM H2O2 at room temperature. Cells were then first washed with quencher solution (1 mM sodium azide, 10 mM sodium ascorbate and 5 mM Trolox in DPBS), then with DPBS, scraped and harvested. Proteinase K digest: All steps were carried out at 4°C unless stated otherwise. Cells were washed and suspended in homogenization buffer I (10 mM KCl, 1.5 mM MgCl2, 10 mM HEPES-KOH and 1 mM DTT pH 7.5). After 20 min of incubation in an overhead shaker, cells were dounced with a tight-fitting pestle and mixed with homogenization buffer II (375 mM KCl, 22.5 mM MgCl2, 220 mM HEPES-KOH and 0.5 mM DTT pH 7.5) at a ratio 1:5 (homogenization buffer I:II). Cleared lysates were obtained by centrifugation at 600xg for 10 min. Samples were treated with 100 µg/ml Proteinase K for 1 h at 37°C and for control samples 0.1% RAPIGestTM was additionally added. Digested material was separated from membrane-protected material by centrifugation at 17,000xg for 15 min. Streptavidin-pulldown: APEX2-labeled pellets after proteinase K digestion were suspended in RIPA buffer containing quenching components (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1x protease inhibitors (Roche), 1x PhosStop (Roche), 1 mM sodium azide, 10 mM sodium ascorbate and 1 mM Trolox), briefly sonified and cleared by centrifugation at 10,000xg. Supernatants were then incubated over-night on pre-equilibrated Streptavidin-Agarose (Sigma). Subsequently, samples were washed 3x in RIPA buffer with quenching components and 3x in 3 M Urea buffer (in 50 mM NH4HCO3) prior to incubation with TCEP (5 mM final) for 30 min at 55°C and shaking. Samples were alkylated with IAA (10 mM final) for 20 min at room temperature, quenched by addition of DTT (20 mM final) followed by 2 washes with 2 M Urea buffer (in 50 mM NH4HCO3) and over-night trypsin digestion with 1 µg trypsin per 20 µl beads at 37°C. Supernatants were collected from the resin plus two additional washes with 2M Urea buffer, acidified with trifluoroacetic acid (1% final) and their volume decreased by vacuum centrifugation. Digested peptides were desalted on custom-made C18 stage tips and reconstituted with 0.5% acetic acid for MS analysis.