Affinity purification coupled with mass spectrometry proteomics

APEX2-mediated biotinylation of cells was carried out as described before (Hung et al., 2016). In brief, 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. 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 mMDTT pH 7.5) at a ratio 1:5 (homogenization buffer I:II). Cleared lysates were obtained by centrifugation at 600xg for 10 min. For mass spectrometry, 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. The resulting pellets were suspended in RIPA buffer containing quenching components (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X, 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. In-solution digest coupled to mass spectrometry was performed as described before (Lobingier et al., 2017). Briefly, 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 (5mM 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. Lyso-IP was performed similar as described before (Abu-Remaileh et al., 2017). In brief, TMEM192-HA and TMEM192-FLAG cells were grown in the presence of BafA1 (200 nM, 2 h), washed twice in PBS, scraped off and transferred to tubes on ice. All proceeding steps were carried out at 4°C unless stated otherwise and all buffers were supplemented with protease inhibitors. After centrifugation, cells were suspended in LysoIP buffer (50 mM KCl, 100 mM KH2PO4 and 100 mM K2HPO4 pH 7.2) and dounce homogenized. Nuclei and unbroken cells were pelleted at 1500xg for 10 min and the supernatant was transferred to fresh tubes and supplemented with 40 µl pre-equilibrated HA-magnetic beads (ThermoFisher). After 1 h rotating incubation, the beads were washed 3x in salt buffer (LysoIP buffer with 300 mM NaCl) and eluted by suspension of the beads in 150 µl urea buffer (8M Urea, 50 mM Tris pH8, 150 mM NaCl) followed by 30 min rotating incubation. The eluate was sonicated for 5 min and sequentially treated with 5 mM DTT (Sigma, 30 min, 55°C and shaking),15 mM IAA (Sigma, 30 min, room temperature) and 10 mM DTT (15 min at room temperature). Proteins were precipitated by adding 600 µl methanol (Roth) and 150 µl chloroform (Merck), vortexing and adding 450 µl of MS-grade H2O (Roth) and vortexing again. After centrifugation at 14,000xg for 5 min, the hydrophilic phase was removed. 450 µl Methanol were added and again spun down at 14,000xg for 5 min. Samples were completely dried by vacuum centrifugation at 30°C and proteins were suspended in 30 µl 50 mM ABC buffer followed by digestion with 1 µg trypsin overnight at 37°C. Digestion was stopped by adding 30 µl 50% acetonitrile (Roth) / 5% formic acid (Merck) for 10 min. Subsequently, all liquids were evaporated by vacuum centrifugation. Peptides were suspended in 30 µl 5% acetonitrile / 1% TFA (Honeywell Fluka).