Affinity purification coupled with mass spectrometry proteomics

Cells were incubated with 500 µM Biotin-Phenol during the last 30 min of LLOMe or GPN 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 RIPA (50mM Tris, 150mM 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 10,000 xg for 10 min before application to streptavidin agarose resin (Thermo) and incubation with overhead shaking overnight. Subsequently, samples were washed 3x in RIPA buffer and 3x in 3 M Urea buffer (in 50 mM ABC) followed by incubation with TCEP (5 mM final) for 30 min at 55°C with orbital shaking. After alkylation with IAA (10 mM final) for 20 min at room temperature in the dark, the reaction was quenched with DTT (20 mM final). Samples were washed 2x with 2M Urea (in 50 mM ABC) before trypsin digestion overnight at 37°C (20 µg/ml final). The resin was spun down and supernatants containing digested peptides were collected. After washing the resin 2x with 2 M Urea and pooling all supernatants the samples were acidified with TFA (1% final). Digested peptides were desalted on custom-made C18 stage tips. Using an Easy-nLC1200 liquid chromatography, peptides were loaded onto 75 µm x 15 cm fused silica capillaries (New Objective) packed with C18AQ resin (Reprosil- Pur 120, 1.9 µm, Dr. Maisch HPLC). Peptide mixtures were separated using a gradient of 5%–33% acetonitrile in 0.1% acetic acid over 35 min and detected on an Orbitrap Elite mass spectrometer (Thermo Scientific). Dynamic exclusion was enabled for 30 s and singly charged species or species for which a charge could not be assigned were rejected.