Search settings were: precursor mass tolerance, 0.05 Da; fragment mass tolerance, 0.05 Da; maximum missed cleavages, 4; variable modifications, acetylation of lysine, methylation of arginine and lysine, and ubiquitination of lysine; enzyme, trypsin.
Search parameters for both algorithms were: a maximum of two missed cleavages, with precursor and fragment mass tolerance set to 20 ppm and 0.3 Da, respectively.
The SwissProt 51.6 (protein) database was searched with following parameters: Rodentia database, monoisotopic tryptic peptides only, peptide mass tolerance at ±2Da, fragment mass tolerance at ±1.5Da and maximum missed cleavages at 3. A decoy database was also searched to eliminate false-positive proteins.
Peptide mass tolerance was set to ± 100 ppm, fragment mass tolerance to ± 0.5 Da with a maximum of two missed cleavages.
The enzyme selected was trypsin, with a maximum missed cleavage of 2. The peptide mass tolerance and the fragment mass tolerance were selected as ± 0.1 Da and ± 0.3 Da, respectively.
The parameters for the search were monoisotopic peptide mass accuracy of 80 ppm, fragment mass accuracy of 0.7 Da, missed cleavage of maximum one, carbamidomethylation of cysteine, and partial oxidation of methionine.
The peptide mass tolerance was 20 ppm, fragment mass tolerance 0.8 Da, and trypsin specificity was applied with a maximum of 2 missed cleavages.
MALDIM-MS ion search was performed with this set of parameters: (I) taxonomy: Mus musculus, (II) proteolytic enzyme: trypsin, (III) maximum of accepted missed cleavages: 1, (IV) mass value: monoisotopic, (V) peptide mass tolerance 0.8 Da, (VI) fragment mass tolerance: 0.8 Da, and (VII) variable modifications: oxidation of methionine and acrylamide adducts (propionamide) on cysteine.
MS/MS ion search was performed with this set of parameters: (i) taxonomy: Rattus norvegicus, (ii) proteolytic enzyme: trypsin, (iii) maximum of accepted missed cleavages: 1, (iv) mass value: monoisotopic, (v) peptide mass tolerance 0.8 Da, (vi) fragment mass tolerance: 0.8 Da and (vii) variable modifications: oxidation of methionine and acrylamide adducts (propionamide) on cysteine.
The Andromeda search engine was used to search the MS/MS spectra using the following parameters: trypsin/P with maximum 2 missed cleavages sites; peptide mass tolerance at first search was set at 20 ppm; MS/MS fragment mass tolerance at 0.49 Da, and top 6 MS/MS peaks per 100 Da and a minimum peptide length of 6 amino acids were required.
