The fixed modification of carbamidomethyl-C was specified, and the included variable modifications were acetylation of the N-terminus, deamidation of N, deamidation of Q and oxidation of M. Components were typically clustered with a 10ppm mass precision and a 0.25 min time tolerance against the database-generated theoretical peptide ion masses with a minimum of one matched peptide.
The search parameters were set as follows: the same fixed and variable modifications as for searching the DDA data, one possible missed tryptic cleavage site, automatic settings for precursor and product ion tolerance, a minimum of 1 peptide matches per protein, with a minimum of 5 consecutive fragment ions per peptide, and a minimum number of product ion matches per protein, 7.
For peptide matching, a minimum of four peptides matches, a maximum of one miscleavage, and peptide modifications by carbamidomethylcysteine and methionine oxidation were accepted.
A valid hit counted as the one with a minimum of 80 nt matches in the alignment.
Protein identification was considered as unambiguous when a minimum of two peptides matched with a minimum score of 20.
For peptide matching, a minimum of four peptides matches and 15% sequence coverage, a maximum of one miscleavage, and peptide modifications by carboxyamidomethylcysteine, methionine sulfoxide, and pyro-glutamic acid or acetylated N-terminal residue, were accepted.
For peptide matching, a maximum of one missed cleavage was allowed and a minimum of four peptide matches was required.
Similarly, MegaBLAST requires a minimum of 28 contiguous matches to generate an alignment.
The parameters for the database search were as follows: automatic tolerance for precursor ions, automatic tolerance for product ions, minimum of three fragment ions matched per peptide, minimum of seven fragment ions matched per protein, minimum of two peptides matched per protein, one missed cleavage, and carbamidomethylation and oxidation of methionine as modifications.
Only managers in charge for a minimum of 50 competitive matches are counted.
