Affinity purification coupled with mass spectrometry proteomics

HeLa cells stably expressing UBE2QL1-APEX2 were grown in lysine- and arginine-free DMEM supplemented with FBS, L-Glutamine, Sodium pyruvate, heavy arginine (R10) (38 μg/ml) and lysine (K8) (66 μg/ml) or light arginine (R0) (38 μg/ml) and lysine (K0) (66 μg/ml), respectively. Further experiments were conducted as soon as the cells reached a protein labelling with heavy amino acids of at least 95%. Heavy-labeled cells were treated with 250 μM Leu-Leu methyl ester hydrobromide (LLOMe, Sigma) for 3 h at 37°C, while light-labelled cells were treated with vehicle alone (EtOH). Proximity labeling was performed in SILAC-labelled HeLa cells stably expressing UBE2QL1-APEX2 as described before. Briefly, cells were incubated with 500 µM Biotin-Phenol during the last 30 min of LLOMe treatment and subsequently pulsed by addition of H2O2 for 1 min at room temperature. To stop the biotinylation reaction, they were washed 3x with quencher solution (10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox in DPBS) and 3x with PBS. All further steps were performed at 4°C unless indicated otherwise. After cell harvest with 0.25% Trypsin/EDTA (ThermoFisher Scientific), cells were counted and heavy- and light-labelled cells were mixed at a 1:1 ratio based on total cell numbers. After centrifugation, the resulting cell pellets were lysed in APEX-RIPA (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) supplemented with 10 mM sodium ascorbate, 1 mM sodium azide, 1 mM Trolox and protease inhibitors (Roche Complete). Samples were sonicated 2x for 1 s, spun down at 10,000xg for 10 min before application to streptavidin agarose resin (ThermoFisher Scientific) and incubation with overhead shaking overnight.