C18 ultramicrospin columns

The expression of SH-tagged bait proteins stably integrated in T-REx-HEK293 Flp–In cells was induced with 1 µg/ml doxycycline for 24 h. For affinity purification, four confluent 150 mm tissue culture plates were harvested and the cell pellet was snap-frozen. Then the cell pellet was lysed in 4 ml lysis buffer (0.5% NP40, 50 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM NaF, 400 nM Na3VO4 supplemented with 1 mM PMSF, 1.2 µM Avidin, and protease inhibitor cocktail (P8849, Sigma)). The cleared cell lysate was incubated with Strep-Tactin beads (IBA LifeSciences) for 1 h on a rotation shaker. Upon washing two times with lysis buffer and three times with the same buffer but without protease inhibitor and detergent, the protein complexes were eluted from the Strep-Tactin beads with 2 mM biotin. Proteins of the eluate were precipitated with trichloroacetic acid and then dissolved in 8 M urea. Cysteine bonds were reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 10 mM iodoacetamide. The proteins were digested with 0.8 µg trypsin (V5112, Promega) over night followed by peptide clean-up with C18 UltraMicroSpin columns (The Nest Group). The dried peptides were dissolved in 2% acetonitrile and 0.1% formic acid.

Peptides were desalted using C18 Ultra Micro Spin columns (The Nest Group) according to the manufacturer’s instructions and dried down using a SpeedVac system. Peptides were resuspended in 17 μL LC solvent A (1% acetonitrile, 0.1% formic acid (FA)) and spiked with the Biognosys iRT kit calibration peptides. Peptide concentrations were determined using a UV/VIS Spectrometer (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany).