After affinity purification, protein samples were partially air-dried in a SpeedVac concentrator and then reconstituted in 1× final Laemmli buffer containing 25 mM dithiothreitol and heated at 95°C for 5 min. Cysteines were alkylated for 30 min at room temperature by the addition of a solution of 90 mM iodoacetamide. Protein samples were loaded on a one-dimensional acrylamide gel (stacking 4%, separating 12%) and the electrophoresis was stopped as soon as the protein sample entered the separation gel. The gel was briefly stained with Coomassie blue (Instant Blue, Expedeon), and a single slice containing the whole sample was excised. The gel slice was washed twice with 100 mM ammonium bicarbonate and once with 100 mM ammonium bicarbonate-acetonitrile (1:1). Proteins were in-gel–digested using 0.6 μg of modified sequencing-grade trypsin (Promega) in 50 mM ammonium bicarbonate overnight at 37°C. The resulting peptides were extracted from the gel by one round of incubation (15 min, 37°C) in 50 mM ammonium bicarbonate and two rounds of incubation (15 min each, 37°C) in 10% formic acid–acetonitrile (1:1). The extracted fractions were pooled with the initial digestion supernatant and dried in a speed-vac. Peptides were further purified by C18 zip-tip (Millipore) and analyzed by nano-LC coupled to either an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) (for samples of biological replicates 1 to 3) or a Q-Exactive mass spectrometer (Thermo Fisher Scientific) (for samples of biological replicate 4). Five microliters of each sample was loaded on a C-18 precolumn (300 μm inner diameter × 5 mm; Dionex) in a solvent made of 5% acetonitrile and 0.05% trifluoroacetic acid at a flow rate of 20 μl/min. After 5 min of desalting, the precolumn was switched online with the analytical C-18 column (75 μm inner diameter × 50 cm; Reprosil C18) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were eluted using a 5 to 50% gradient of solvent B over 105 min at a flow rate of 300 nl/min. The mass spectrometer was operated in a data-dependent acquisition mode with Xcalibur software. On the LTQ-Velos, survey MS scans were acquired in the Orbitrap on the 350 to 1800 m/z range with the resolution set at 60,000 and automatic gain control (AGC) target at 1 × 106 ions, the 20 most intense ions were selected for collision-induced dissociation, and MS/MS spectra were acquired in the linear trap with an AGC target at 5 × 103 ions, maximum fill time at 100 ms, and a dynamic exclusion of 60 s to prevent repetitive selection of the same peptide. On the Q-Exactive, survey MS scans were acquired on the 350 to 1500 m/z range with the resolution set at 70,000 and AGC target at 3 × 106 ions, the 10 most intense ions were selected for higher-energy C-trap dissociation, and MS/MS spectra were acquired in Orbitrap with an AGC target at 1 × 105 ions, maximum fill time at 100 ms, and a dynamic exclusion of 30 s. Triplicate or duplicate technical LC-MS measurements were performed for each sample.

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