Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii Joseph M. Dybas1,2,3, Carlos J. Madrid-Aliste1,2,3, Fa-Yun Che1,5,8, Edward Nieves1,5,8, Dmitry Rykunov1,2,3, Ruth Hogue Angeletti1,3,5,8, Louis M. Weiss1,4,6, Kami Kim1,6,7, Andras Fiser1,2,3* 1 Biodefense Proteomics Research Center, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 3 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, United States of America, 4 Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 5 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 6 Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, United States of America, 7 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 8 Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, New York, United States of America Abstract 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. Citation: Dybas JM, Madrid-Aliste CJ, Che F-Y, Nieves E, Rykunov D, et al. (2008) Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii. PLoS ONE 3(12): e3899. doi:10.1371/journal.pone.0003899 Editor: Steven L. Salzberg, University of Maryland, United States of America Received September 10, 2008; Accepted November 7, 2008; Published December 9, 2008 Copyright: 2008 Dybas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Financial support was provided by NIH-NIAID HHSN266200400054C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: afiser@aecom.yu.edu Introduction Toxoplasma gondii is an obligate intracellular protozoan, belong- ing to the phylum Apicomplexa and is an important pathogen in both immune competent and immune compromised humans. The parasite causes chronic infection in adults and is present in an estimated 22.5% of people older than 12 in the United States [1] and up to 90% of the population in other regions of the world [2]. Acute infection is typically not symptomatic. Reactivation of latent infections is seen in immune compromised individuals, where infection often presents as encephalitis. T. gondii clinical disease is most typical in immune compromised individuals and is a common opportunistic pathogen associated with AIDS. T. gondii has a wide range of hosts including almost all mammals as well as birds. It exists in three life stages. Oocysts are produced in the definitive host, the cat, and are environmentally resistant, surviving for prolonged periods of time in water, thus causing potential waterborne illness. Bradyzoites, found in the intermediate hosts, are slow growing parasites contained in vacuoles, which form tissue cysts and are generally unrecognized by the host’s immune system. Bradyzoite tissue cysts can be transmitted via ingestion of undercooked, infected meat products or contaminated water. Once the parasite is present in the host, bradyzoites or sporozoites differentiate into the tachyzoite stage, which is responsible for the dissemination and clinically apparent infection. Due to waterborne outbreaks associated with ocular toxoplasmosis [3], T. gondii is classified by the National Institute of Allergy and Infectious Diseases as a Category B priority pathogen. T. gondii is an important model system for the phylum Apicomplexa [4], which includes, among others, Plasmodium (malaria) and Cryptosporidium species. Unlike many other Apicom- plexa, which are experimentally intractable, T. gondii is easily cultured in vitro, has well established experimental protocols for genetic manipulation, and has a well characterized mouse model [5]. PLoS ONE | www.plosone.org 1 December 2008 | Volume 3 | Issue 12 | e3899