To elute cys-nitrosylated peptides that remained covalently attached to the beads after on-bead digestion of SNO-Ps, 100 μl of buffer B were added to each sample, followed by the addition of 10 μl of 100 mM DTT (incubation for 1 hour at 37°C). Alkylation of released cysteines was achieved by adding 10 μl of 200 mM iodoacetamide (for 1 hour at 37°C in the dark). The supernatants were recovered, and the beads were washed with 50 μl of buffer B. Supernatants and their respective washes were pooled and evaporated in a vacuum centrifuge (samples: SNO peptides). For a qualitative analysis of putative nitrosylation sites, SNO peptides from the evaporated extracts were dissolved in 200 μl of solution SCX-a, purified by SCX StageTips as described, evaporated to dryness, and reconstituted in 10 μl of 0.1% formic acid/2% acetonitrile. Five microliters of aliquot of the pooled sample was subjected to nanoLC-MS/MS analysis. Peptides detected in the CysNO data set of experiment 5 were assigned to proteins present in the SNO-P data set (biological n of ≥3). Notably, shared peptides that were detected under both Cys and CysNO conditions were also included in our combined data set (table S13).

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