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Background    Toxoplasma gondii  is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromized hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the  T. gondii  proteome.    Methodology/Principal Findings  We have explored the proteome of  T. gondii  tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the  T. gondii  proteome. The proteomics survey identified 609 proteins that are unique to  Toxoplasma  as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%.    Conclusions/Significance  This study not only provides the largest proteomics exploration of the  T. gondii  proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes.

Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii