Affinity purification coupled with mass spectrometry proteomics

Anti-HA-immunoprecipitation was performed as previously described (Behrends et al, 2010; Jung et al, 2015; Jung et al, 2017; Sowa et al, 2009). Summarily, expression of TAPL-HA and coreTAPL-HA was induced by addition of 4 µg/ml doxycycline for 24 h in HeLa Flp-In T-REx cells. Parental non-transfected HeLa Flp-In T-REx cells were used as negative control. For each sample, 6.4 x 107 cells were harvested, frozen in liquid nitrogen and stored at -80 °C. Cells were lysed in 3 ml MCLB buffer (50 mM Tris, 150 mM NaCl, 0.5% NP40, pH 7.4) supplemented with cOmplete EDTA-free protease inhibitor tablets (Roche, 2 tablets for 50 ml MCLB buffer) on ice for 30 min. Lysate was cleared by centrifugation (18,000x g, 10 min, 4 °C) followed by filtering through a 0.45 µm spin filter (Millipore). For isolation of HA-tagged proteins, cell lysate was incubated overnight at 4 °C with 60 µl of equilibrated α-HA agarose beads (Sigma-Aldrich/Merck). Subsequently, beads were washed four times with 1 ml MCLB and 1 ml PBS, respectively. 50 µl of 250 µg/ml HA peptide was added to dry beads and incubated for 30 min at room temperature. Elution was repeated twice obtaining a final volume of 150 µl. Proteins were precipitated with 20% tri-chloroacetic acid (TCA), resuspended in 20 µl 50 mM ammonium bicarbonate pH 8.0 containing 10% acetonitrile and 750 ng trypsin (Promega) and incubated for 4 h at 37 °C. Desalting was performed using stage tips. Samples were analyzed as technical duplicates on an LTQ Velos (ThermoScientific).