Immunoprecipitation buffer

Cell-surface proteins were purified using the EZ-Link Sulfo-NHS-SS-Biotin system (Thermo Fisher Scientific). Cells were rinsed with PBS ( + CaCl₂/MgCl₂) and incubated in a 0.5 mg ml⁻¹ biotin solution for 1 h on ice. The free biotin was quenched by three 5-min washes with a 50 mM glycine solution. Cells were rinsed with PBS and lyzed in an immunoprecipitation buffer (Cell Signaling Technology) containing protease inhibitors. After centrifugation of the lysate, Neutravidin beads (Thermo Fisher Scientific) were added 1:2 to the supernatant. The beads/lysate mixture was incubated at 4 °C for 3 h under constant rotation. Beads were washed once with the immunoprecipitation buffer and then three times for 5 min with a 50 mM Tris-HCl solution. After on-bead digestion with trypsin, purified peptides were labeled and analyzed by tandem mass spectrometry using a 180-min MS3 method on an Orbitrap Fusion Lumos mass spectrometer.

Harvested cells were lysed using ultrasonicator for three times 1 min at 0.6 cycle and 90% amplitude and proteins were extracted using 50 mm ammonium bicarbonate buffer containing 8 m urea, protease inhibitors, and 50 μm deubiquitinase inhibitor PR619. For each sample, 20 mg protein were reduced and alkylated using 5 mm DTT and 10 mm chloroacetamide, respectively. Subsequently, samples were digested with lys-C (1:50 w/w enzyme:protein ratio). After buffer dilution (to 2 m urea), samples were digested with trypsin (1:50 w/w enzyme:protein ratio). The peptide product was then purified using a Seppak C8 column and concentrated using a speedvac. Finally, the purified peptides were reconstituted in the immunoprecipitation buffer for further enrichment by immunoprecipiation with an antibody recognizing the diglycyl-remnant. The immunoprecipitation buffer was supplied by Cell Signaling Technology as part of the enrichment kit. Details on extraction, digestion, and peptide purification were described previously (26 (link), 27 (link)).