[32], a combined transmembrane topology and signal peptide predictor, was used to perform the transmembrane domain and signal peptide predictions. There are 13,430 sequences (45% of the combined dataset) with at least one PFAM domain annotation compared to 20,326 sequences (67%) that are annotated with orthologs predicted from pairwise BLAST [22] alignments with sequences from the NCBI NR database. The percentage of proteins that have PFAM domains compares well with the published results of PFAM 22.0, which reports that 49% of 213 T. gondii UniProtKB/TrEMBL deposited proteins have at least one PFAM domain [31]. Sequences with PFAM domains correspond to 5,741 clusters (38% of the total clusters). The TigrScan, TwinScan, and Release4 sequence sets have similar percentages of sequences with PFAM domains (36%, 43%, and 44%, respectively). The percentages of sequences with PFAM domains are similar for the Glimmer and NR sequence sets (62% and 69%, respectively). The higher proportion of PFAM domains found in the NR sequences, compared to TigrScan, TwinScan, or Release4, is probably a consequence of a higher level of confidence in these sequences, as they are typically experimentally derived. However, the higher proportion of PFAM domains for the Glimmer sequences is likely a consequence of the fact that they are much longer, on average, than the other sequence types (Table S1), so an individual sequence will coincide with a bona fide coding region within the genome with a higher probability than the other protein types. In summary, PFAM annotations are used to infer a certain degree of biological validity for approximately 45% of the compiled T. gondii proteome. There are 6,927 sequences (23% of the combined dataset) that have predicted transmembrane domains, which corresponds to 3,730 clusters (25% of the total clusters), and which is consistent with the proportion of membrane proteins that have been predicted in other genomes (20–35%) [33]. There are 4,330 sequences (14% of the combined dataset) that have predicted signal peptides, which corresponds to 2,601 clusters (17% of the total clusters). The percentages of sequences with signal peptides and transmembrane regions are consistent among the individual datasets. Transmembrane proteins of T. gondii are of particular interest because of their role in the interaction and adhesion of the parasite to the host cell and their link to the virulence of the parasite. These critical functions make some subsets of mem- brane proteins good candidates for chemotherapeutic targets. Meanwhile sequences with signal peptides generally refer to proteins that are secreted from the cell. In the case of T. gondii, secreted proteins include those of the micronemes, rhoptries, and dense granules, which are integral parts of the unique process of interaction, invasion, and infection of the host cell by T. gondii [34]. Comparative genomics of the T. gondii proteome Each sequence was compared against other Apicomplexan genomes, against the human genome, and against the complete NCBI NR database (Table S1). The amino acid sequences of the hypothetical proteome of T. gondii were searched against the complete NCBI NR database, Apicomplexa proteins, and human proteins to identify unique and conserved proteins. In general, 67% of the hypothetical T .gondii proteome (59% of the clusters) has a homologous sequence in NR and 64% has an Apicomplexan ortholog (58% of the clusters). Approximately 52% of T. gondii sequences (57.5% of the clusters) are unique as compared to the human genome and, thus, fulfill a primary requirement of chemotherapeutic target candidates. Validation of Gene Predictions EPIC-DB: Experimental ProteomICs DataBase All experimental data, annotations, protein cluster information and comparative genomics data are organized into a relational database (EPICDB) that is publicly accessible at http://www. fiserlab.org/epicdb. In addition, a summary of the data and information about reagents generated from the Toxoplasma gondii MS experiments are available from the Resource Center for Biodefense Proteomics Research website http://www.proteomics- resource.org/. Discussion The proteomics study presented here is one of the largest of a genome and certainly the most comprehensive analysis and validation of the T. gondii proteome to date identifying novel proteins and offering unique insights into its gene structure. We experimentally validated with MS experiments 6,438 distinct proteins that can be clustered into 2,477 groups and account for approximately one third of the T. gondii genome. Further, this analysis provides important novel information, with respect to the T. gondii proteome, since 3,838 (60%) of the experimentally identified proteins have been annotated as ‘‘hypothetical’’, ‘‘putative’’ or ‘‘predicted’’ in the NCBI NR database. Additionally 609 MS identified T. gondii proteins are unique as compared to any known organisms, providing an important subset of validated proteins for drug targeting. While the genomes of many organisms have been sequenced, there are relatively limited amounts of experimental protein data to accurately annotate these genomes or to validate the computational gene prediction methods. The current study offers a way to address this ubiquitous problem by integrating genomics and proteomics data. The large scale experimental validation of the hypothetical T. gondii genome shows a false negative rate for various gene prediction methods of about 31–42% (proteins that were confirmed experimentally by both MS and EST data but missed by one or more of the computational gene prediction methods) and experimentally confirms earlier anecdotal reports that gene prediction algorithms operate with a substantial false negative rate [29,30]. This study demonstrates that genome analysis coupled with experimental proteomics data is essential to benchmark and improve the accuracy of gene prediction methods and facilitates insights into gene structure and splice variability within genomes. Materials and Methods Protein sequence datasets All available T. gondii protein sequences were compiled from five datasets; TigrScan (8,336 sequences), TwinScan (7,588), Glim- merHMM (4,954), Release4 (7,793), and NR sequences (1,526). TigrScan, TwinScan and GlimmerHMM sequences were down- loaded as FASTA files of protein sequences from http://www. toxodb.org/common/downloads/release-3.3/Genome/pep/. A FASTA file of Release4 sequences was downloaded from http:// www.toxodb.org/common/downloads/release-4.0/TgondiiAnno- tatedProteins.fa. The NR sequence dataset was obtained from the NCBI Entrez Protein Database, which was filtered, by the organism name, for Toxoplasma gondii (As of July 2008, predicted genes/ proteins from T. gondii genome analysis have not been deposited to NCBI or GenBank databases). The five datasets were combined to provide a comprehensive set of all available protein sequences for the Toxoplasma gondii proteome and comprised 30,197 sequences. This is a redundant database as different prediction methods may identify the same sequence with different names. PLoS ONE | www.plosone.org 8 December 2008 | Volume 3 | Issue 12 | e3899