Neuronal nuclear protein extracts (50 μg) were precipitated by adding cold acetone [4:1 (v/v)] and incubating the solution for 1 hour at −20°C. The pellet was solubilized in 50 μl of 5% sodium DOC containing 60 mM tris buffer (pH 8.8). Cysteine alkylation was achieved by adding sequential aliquots of, respectively, 5 μl of 100 mM DTT (1-hour incubation at 37°C), 6 μl of 200 mM iodoacetamide (1-hour incubation at 37°C), and 1 μl of 100 mM DTT (20-min incubation at 37°C). Solution volume was brought to 500 μl by adding 20 mM tris buffer. Overnight protein digestion was carried out at 37°C after adding sequencing-grade trypsin [1:100 (w/w); Sigma-Aldrich]. The enzymatic reaction was quenched by the addition of 50 μl of 5% trifluroacetic acid, which served also for precipitating the DOC detergent. After spinning down the precipitate, 200 μl of the supernatant was removed and purified on RP StageTips, as described in previous paragraphs. The purified peptide mixture was fractionated on SCX StageTips, as described above. Stepwise elution of tryptic peptides from SCX StageTips was achieved by sequential addition of 20 μl of seven solutions, all containing 20% acetonitrile, of increasing ionic strength and pH: (i) 50 mM ammonium acetate and 0.5% acetic acid, (ii) 75 mM ammonium acetate and 0.5% acetic acid, (iii) 100 mM ammonium acetate and 0.5% acetic acid, (iv) 150 mM ammonium acetate and 0.5% acetic acid, (v) 250 mM ammonium acetate and 0.5% acetic acid, (vi) 350 mM ammonium acetate and 0.5% acetic acid, and (vii) 500 mM ammonium acetate. Fractions were evaporated by vacuum centrifugation and resuspended in 15 μl of 0.1% formic acid/2% acetonitrile. Two microliters of aliquot of each fraction was subjected to nanoLC-MS/MS analysis using a long elution gradient (see below).

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