Mascot search analysis parameters included: trypsin enzyme specificity, allowance for 2 missed cleavages, peptide mass tolerance of 20 ppm for precursor ions and fragment mass tolerance of 0.8 Da.
The resulting mass data was interrogated using an offline version of the MASCOT search engine (Matrix Science) using the SPtrEMBL database with the following search criteria: allowance of 0 or 1 missed cleavages; peptide mass tolerance of 50 ppm; trypsin as digestion enzyme; carbamidomethyl modification of cysteine; methionine oxidation as partial modification; and charged state as MH+ [ 14].
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.
Protein searches were performed with the mouse protein database downloaded from the SWISS-PROT website (http://www.ebi.ac.uk/uniprot) with the following parameters: an allowance for two possible missed cleavage sites, a peptide mass tolerance of 20 ppm, and a fragment mass tolerance of 0.50 Da.
For MS/MS data, the peptide mass tolerance was 0.5 Da, MS/MS ion mass tolerance at 0.5 Da, allowance of 1 missed cleavage, and charge state +1.
The following mass search parameters were set: peptide mass tolerance, 50 ppm; a mass window between 0 and 100 kDa, allowance of missed cleavage, 2; consideration for variable modifications such as oxidation of methionine and propionamides of cysteines.
Searching parameters were set as following: enzyme, trypsin; allowance of up to one missed cleavage peptide; the peptide mass tolerance,1.0 Da and the fragment ion mass tolerance, 0.3 Da; fixed modification parameter, carbamoylmethylation; variable modification parameters, oxidation; auto hits allowed; results format as peptide summary report.
A mass tolerance of 0.3 Da for both parent (MS) and fragmented (MS/MS) ions, allowance for up to one trypsin miscleavage, variable amino acid modifications consisting of methionine oxidation and cysteine carbamidomethylation were used.
For protein identification, a mass tolerance of ±0.05 Da was permitted for intact peptide masses and ±0.1 Da for fragmented ions, with allowance for one missed cleavages in the trypsin digests.
