Protein identification and quantification were achieved in Proteome Discoverer 1.4 (Thermo Fisher Scientific) using Sequest as the search engine and the Rattus proteome sequence database (www.ebi.ac.uk/uniprot; accessed on March 2015). The database was merged with a list of common contaminants named “Common Repository of Adventitious Proteins” retrieved from the Global Proteome Machine website (www.thegpm.org/crap/index.html). In total, 27,927 entries were searched. The following general search parameters were used: MS tolerance, 15 parts per million; MS/MS tolerance, 0.02 Da; enzyme, trypsin; maximum missed cleavages, 2; variable modification, oxidized methionine. In addition, the following search parameters were used depending on the experiment: experiments 1 and 2: variable modification, C-terminal 18O labeling; fixed modification, MMTS; experiments 3 and 4: variable modifications, NEM and carbamidomethyl-cysteine (CAM); fixed modifications (in two parallel searches), (i) N-terminal dimethyl (light) and dimethyl-lysine (light) and (ii) N-terminal dimethyl (medium) and dimethyl-lysine (medium); experiment 5: variable modification, C-terminal 18O labeling; fixed modification, NEM. For SNO-peptide searches, the following additional parameters were used: experiments 1 and 5: fixed modification, CAM. False discovery rate estimation was performed in Proteome Discoverer using the Target-decoy validator node. Minimum requirement for protein ID for every experiment was two peptides at 95% confidence, whereas for SNO-peptide identification, the peptide confidence threshold was set to 99%.

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