Proteins from each pixel were identified from the MS/MS spectra using the SEQUEST Browser program (Thermo Electron). DTA files were generated from MS/MS spectra using an intensity threshold of 500 000 (Deca XP+ data) or 5000 (LTQ data), and minimum ion count of 30. The DTA files generated were processed by the ZSA, CorrectIon, and IonQuest algorithms ofthe SEQUEST Browser program, and searched against the International Protein Index (IPI) human protein database  version 2.21 (July, 2003) containing 56 530entries as requested by HUPO. In some of the optimization experiments (data sets for Fig. 2A), the National Center for Biotechnology Information nonredundant database (01/15/ 2004) was also used. To reduce database search time, the databases were indexed with the following parameters: average mass range of 500-3500, length of 6-100, tryptic cleavages with 1 (for LTQ analysis) or 2 (for LCQ Deca XP + analysis) inter nal missed cleavage sites, static modification of Cys by carboxamidomethylation, and dynamic modification of Met to methionine sulfoxide (+16 Da). The DTA files were searched with a 2.5 Da peptide mass tolerance and 0 Da fragment ion mass tolerance. Other search parameters were identical to those used for database indexing. For each pixel, the peptides identified were assembled into the minimum number of unique proteins using SEQUEST SUMMARY with a depth of 3. Perl programs were developed for parsing, storing, analyzing, and retrieving SEQUEST results. Data from SEQUEST SUMMARY were stored in a relational database (Oracle 9i) with Perl Object layer.
The peptides from each protein were initially filtered using the following criteria: XCorr > 1.9 (z = 1), 2.3 (z = 2), 3.75 (z = 3) and DCn > 0.1; or Sf > 0.7. Further data analysis used the HUPO defined criteria where peptides were filtered using XCorr > 1.9 (z = 1), 2.2 (z = 2), 3.75 (z = 3) and DCn > 0.1; and RSp < 4. For both criteria, redundant peptides with the same accession number were removed and different forms (charge states and modification) ofthe same peptide were counted as a single-peptide hit. Keratins were also excluded from all data sets.
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