Affinity purification coupled with mass spectrometry proteomics

Cells (4x 15 cm dishes) were harvested and lysed with 3 ml lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Nonidet P40 (NP40) and EDTA-free protease inhibitor cocktail tablets). Centrifugation-cleared lysates (13,000 rpm) were filtered through 0.45 µm spin filters (Millipore Ultrafree-CL) and immunoprecipitated with 60 µl anti-HA resin. Resin containing immune complexes were washed five times with lysis buffer followed by five PBS washes, and elution with 150 µl of 250 mg/ml HA peptide in PBS. Eluted immune complexes were precipitated with 20% trichloroacetic acid (TCA) and pellets were washed once with 10% TCA and four times with cold acetone. Precipitated proteins were resuspended in 50 mM ammonium bicarbonate (pH 8.0) with 20% acetonitrile and incubated with sequencing grade trypsin at a concentration of 12.5 ng/ml at 37ºC for 4 h. Trypsin reactions were quenched by addition of 5% formic acid and peptides were desalted using C18 Stage Tips. For each liquid chromatography coupled to tandem mass spectrometry run using an LTQ Velos linear ion trap mass spectrometer (Thermo Scientific) 4 µl were loaded onto a 18 cm x 125 µm (ID) C18 column and peptides eluted using a 50min 8–26% acetonitrile gradient. Spectra were acquired using a data-dependent Top-10 method. Each sample was shot twice in succession, followed by a wash with 70% acetonitrile and 30% isopropanol.