Affinity purification and sample preparation for liquid chromatography–mass spectrometry (LC–MS/MS)

Cells grown in the “heavy” and “light” media were harvested and lysed in lysis buffer (50 mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 7.5% glycerol; 1% Triton X-100) supplemented with EDTA-free protease inhibitors (Roche, Switzerland). Benzonase was added to minimize nucleic acid contamination. The protein concentration of samples was determined and light and heavy lysates were mixed in a 1:1 ratio. Lysate was incubated with the anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4 °C overnight. The resin was washed with the lysis buffer to remove all nonspecific proteins. The bound FLAG-tag protein along with its interaction partners was eluted from the column with a solution containing FLAG peptide. Eluates were further precipitated by sodium deoxycholate, trichloroacetic acid (TCA), and acetone to remove salts and detergents. Pellets were dissolved in LDS buffer and separated by SDS-PAGE (Invitrogen). Gels after electrophoresis were stained with colloidal blue (Invitrogen) and cut into 10 fractions. In-gel trypsin digestion of the excised protein spots was done. Prior to nanoLC-MS analysis, the samples were desalted using custom-made reversed-phase microcolumns. Peptides were eluted using nonlinear gradient with gradually increasing acetonitrile (ACN) content in the range 2–40% (v/v) with 0.1% trifluoroacetic acid (TFA) from the desalting microcolumn⁶⁸. (Fig. 1B).