Oxidation of methionine and N-terminal protein acetylation for variable modifications and cysteine caramidomethylation for fixed modification.
The database searches were performed allowing for fixed modification of cysteine residues (S-carbamidomethylation, +57.0 Da) and variable modification of methionine residues (oxidation, +16.0 Da), peptide mass tolerance ±2.0 Da and fragment m/z tolerance ±0.8.
The peptide modifications were the same for each search (fixed modifications: methylthio for cysteines, iTRAQ 8-plex for lysine and peptide N-terminus and variable modifications: oxidation of methionine).
Carbamidomethylation of cysteine was set as a fixed modification for quantitative proteome analysis, and carbamidomethylation of cysteine and a TMT 6-plex at the N-terminus and lysine were set as fixed modifications for qualitative proteome analysis.
C2D3-acetylation of lysine side chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses.
The default search settings used for protein identification were: MS/MS accuracies were set to < 0.6 Da, and two missed cleavages for full trypsin with fixed modifications Carbamidomethyl (C); variable modifications: deamidation (N, Q); oxidation (M) and propionamide (C).
The respective search parameters for precursor and product ion mass tolerance were ± 40 ppm and ± 0.8 Da, with allowance made for one missed semiTrypsin, fixed modifications of cysteine through carbamidomethylation and variable modification through lysine carbamidomethylation and methionine oxidation.
The following settings were used: enzyme trypsin, allowing for one missed cleavage, fixed modifications were carbamidomethyl (C), variable modifications were set to Desthiobiotin (K), Acetyl (Protein N-term) and Oxidation (M).
The following parameters were used for searches using SEQUEST v.3.3.1 SP1: enzyme, trypsin; fixed modifications, carboxymethyl cysteine; variable modifications, oxidation of methionine; peptide tolerance, ±1.50 amu; fragment ion tolerance, ±0.5 amu; and number of missed cleavage sites, 1.
The search parameters were: Trypsin/P with two missed cleavages, 10 ppm mass tolerance for MS, 0.5 Da tolerance for MS/MS, fixed modification Carbamidomethyl (C), and variable modifications of Acetyl (Protein N-term), Deamidated (NQ), Dioxidation (M), Formyl (N-term), Gln- > pyro-Glu (N-term Q), Methyl (E), and Oxidation (M).
