Purification of biotinylated proteins for mass spectrometry

MDCK II Tet-off cells inducibly expressing Myc–biotin-ligase–occludin (Fredriksson et al., 2015) were cultured in absence of doxycycline for 6 days in 75-mm transwell inserts (Corning) and then treated or not for 48 h with 100 ng/ml IFN and 30 ng/ml TNF; 50 μM biotin (Millipore Sigma, Billerica, MA) was added for the final 16 h. For each proteomic analysis, cells from six filters were combined. Affinity capture of biotinylated proteins was performed as described previously (Van Itallie et al., 2013) with slight modifications; cells were washed in PBS twice, scraped in PBS, pelleted and lysed in 1.5 ml of radioimmune precipitation buffer (1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.5, 150 mM NaCl supplemented with protease inhibitors). Samples were sonicated, incubated on ice for 20 min, resonicated, and centrifuged for 20 min at 12,000 g to remove insoluble material. Supernatants were transferred to fresh tubes containing 50 μl of (slurry) prewashed Dynabeads MyOne Streptavidin C1 and incubated for 2 h at 4°C in an end-over-end mixer. Beads were washed for 8 min twice with 2% SDS; once with 0.1% deoxycholate, 1% Triton X-100, 500 mm NaCl, 1 mm EDTA, 50 mm Hepes, pH 7.3, and once with 250 mm LiCl, 1 mm EDTA, 0.5% deoxycholate, 0.5% Nonidet P-40, 10 mm Tris-HCl pH 8.0, followed by two washes in 50 mm Tris-HCl pH 7.5, 50 mm NaCl at room temperature. Bound proteins were eluted by a 10-min incubation at 96°C in biotin-saturated 4× SDS sample buffer (8% SDS, 250 mm Tris, pH 6.8, 0.57 m mercaptoethanol, 40% glycerol). Eluted proteins were subjected to SDS-PAGE, and gels were stained briefly with SimplyBlue Safe Stain (Invitrogen). Lanes were excised and divided into 12–16 bands, then destained, reduced, alkylated and digested overnight with trypsin (Promega, V511A Sequencing grade). Eluted peptides were purified on ZipTips (C18, Millipore) and transferred into sample vials (Agilent, Santa Clara, CA) for mass spectrometry.