For the experiments performed on neuronal nuclear extracts treated with either 1 mM Cys or CysNO (table S16), beads were thawed at 4°C, centrifuged at 4000 rpm for 1 min at RT. The supernatant was digested at 37°C with 800 ng of trypsin proteomics grade (Sigma-Aldrich) in 100 μl of 100 mM tris buffer (pH 8.5; buffer A). After tryptic digestion, supernatants were recovered. Beads were washed three times with 20 mM ammonium bicarbonate in 50% MeOH (v/v) (buffer B). Ammonium bicarbonate washes were pooled, dried, and then combined with the first supernatant. Digested proteins samples were subjected to 18O labeling as in (43). Thirty microliters of aliquots of A2, A3, B1, and B2 (table S16) was dried and resuspended in 50 μl of H2O18 98% pure (Sigma-Aldrich)/methanol 4:1 (v/v). The solution was then buffered by tris buffer. A fresh trypsin aliquot (200 ng in 0.5 μl) was added to catalyze the oxygen exchange reaction, which was allowed to proceed overnight at 37°C. Thirty microliters of aliquots of A1, A2, B2, and B3 was dried, resuspended in 30 μl of normal high-performance LC water, and subjected to a parallel trypsin-catalyzed oxygen exchange reaction. Although no change in the molecular weight of control peptides [“light” (L) peptides] was produced, the reaction was allowed to proceed in parallel to the “heavy” (H) labeling reaction to avoid any possible source of bias. After labeling, to inactivate trypsin and avoid the phenomenon of back exchange, samples were heated for 1 hour at 56°C and boiled for 10 min at 100°C. Labeled samples were mixed in a 1:1 ratio as follows: A2(H)/A1(L), A3(H)/A2(L), B2(H)/B3(L), and B1(H)/B2(L). This arrangement allowed the relative comparison of proteomes isolated from CysNO-treated cells versus untreated cells and CysNO-treated cells versus CysNO minus ascorbate-treated cells, in duplicate analysis with forward/reverse labeling. Mixed samples were purified by strong cation exchange (SCX) StageTips (44). Peptide mixtures were diluted with 1.5 ml of 80% acetonitrile/0.5% formic acid (solution SCX-a) and loaded onto a 200-μl micropipette tip stacked with one layer of a SCX resin (Empore extraction disks, Sigma-Aldrich) previously conditioned with 20 μl of solution SCX-b (20% acetonitrile/0.5% formic acid) and 20 μl of solution SCX-a. After two washing steps (20 μl of solution SCX-a and 20 μl of solution SCX-b), elution of tryptic peptides was achieved by adding 7 μl of 500 mM ammonium acetate/20% acetonitrile (solution E). The eluate was dried and resuspended in 15 μl of 0.1% formic acid/2% acetonitrile. Five microliters of aliquot of each sample was subjected to nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) analysis.

For the experiments on neuronal nuclear extracts treated with either 200 μM Cys or CysNO (table S17), beads were thawed at 4°C, centrifuged at 4000 rpm for 1 min at RT. The supernatant was removed and digested at 37°C with 300 ng of trypsin proteomics grade (Sigma-Aldrich) in 100 μl of 100 mM triethylammonium hydrogen carbonate buffer (TEAB) (pH 8.5; buffer C). After tryptic digestion, the supernatants were recovered, and the beads were washed three times with 20 mM TEAB in 50% (v/v) MeOH (buffer D). The washes and the first supernatant of each sample were pooled, dried, and resuspended in 100 μl of buffer C (samples: SNO-Ps). Proteins were reduced further by adding 10 μl of 100 mM DTT (1 hour at 37°C), and residual free cysteines were alkylated by adding 12 μl of 200 mM iodoacetamide. After quenching the reaction with additional 2 μl of 100 mM DTT, complete overnight digestion was achieved by adding a new 300 ng of aliquot of trypsin. Samples were labeled according to the standard dimethyl labeling procedure (45). Briefly, samples were subjected to reductive amination by adding 4 μl of 0.6 M sodium cyanoborohydride and 4 μl of either 4% (w/v) formaldehyde (“light” labeling) or 4% (w/v) formaldehyde-d2 (“medium” labeling). Samples were labeled as light (L) or medium (M) and mixed in pairs (table S18). Digest pairs were diluted to 2.4 ml with SCX-a solution and purified by SCX StageTips, as described above. The six eluates were evaporated, and peptides were resuspended in 12 μl of 0.1% formic acid/2% acetonitrile. Four microliters of aliquot of each sample was subjected to nanoLC-MS/MS analysis with technical duplicates (two injections per sample).

For the experiment on nuclear and cytoplasmic extracts treated with either 200 μM Cys or CysNO (table S19), SNO-Ps were isolated, and digested proteins were prepared and labeled by 18O as described above, with minor modifications in starting amounts of samples subjected to isotopic labeling. The volume of the supernatant and evaporated washes recovered from on-bead digestion was 100 μl. Starting amounts for the heavy labeling reaction were as follows: 45 μl for samples 1 and 2, 25 μl for samples 3 and 5, and 2 × 25 μl for sample 4. The same amount for each of the five samples was taken for the light labeling reaction.

Labeled samples (table S18) were mixed in a 1:1 ratio as follows: 1(H)/2(L), 1(L)/2(H), 4(H)/3(L), and 3(H)/4(L). Mixed samples were purified by SCX StageTips, as described in experiment 1. Because of the higher amount of protein sample recovered in this experiment, sample fractionation at the peptide level could be achieved (four fractions). After tip conditioning, sample loading, and washing, stepwise elution of tryptic peptides was achieved by sequential addition of 7 μl of four solutions, all containing 20% acetonitrile, of increasing ionic strength and pH: (i) 60 mM ammonium acetate and 0.5% formic acid, (ii) 120 mM ammonium acetate and 0.5% formic acid, (iii) 250 mM ammonium acetate and 0.5% formic acid, and (iv) 500 mM ammonium acetate. The four SCX fractions were evaporated by vacuum centrifugation and resuspended in 8 μl of 0.1% formic acid/2% acetonitrile. Six microliters of aliquot of each fraction (four fractions × six H/L pairs) was subjected to nanoLC-MS/MS analysis.

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