Mascot searchingalgorithm

The obtained data files (.raw) were converted into mascot generic files using the MassMatrix File Conversion Tool (Version 2.0; http://www.massmatrix.net) for input into the Mascot searching algorithm (Matrix Science). The data files (all merged in one search) were searched against NCBInr (v. 20080527) with plant taxonomy parameter using the following search criteria: tryptic peptides with up to one missed cleavage and carbamidomethylation of cysteines and oxidation of methionines, which were set as variable modifications. In a separate search, individual bands were searched against an in-house database containing sequences of the recombinant proteins. Results were exported to Microsoft Office Excel â software with a score threshold set at 34; scores above this indicate identity or extensive homology.
The proteins identified in each sample were ranked with the protein with the highest mascot score listed first. The emPAI was also calculated as previously described (Ishihama et al., 2005) . The percentage of the total protein weight contributed by each individual protein was calculated.