directions were grouped into co-localized cluster regions. The genomic regions spanned by the co-localized cluster regions were defined as potential protein coding regions. Growth of T. gondii in vitro RH strain T. gondii were grown in human fibroblasts in DME media with 10% fetal calf serum and 1% Penicillin Streptomycin (GIBCO-BRL) in a 5% CO2 incubator. Parasites were transferred to fresh culture human fibroblast monolayers biweekly. The absence of Mycoplasma contamination was monitored monthly using a PCR method (GIBCO-BRL). Purification of membrane and cytoskeleton protein from T. gondii 1.261010 purified T. gondii RH strain tachyzoites from tissue culture were resuspended in 20 ml of SMDI buffer (250 mM Sucrose, 10 mM MOPS-KOH, pH 7.2, 2 mM DTT, 16protease inhibitor cocktail) and disrupted by French press at a pressure of 1000 PSI, medium setting. The lysate was centrifuged at 7566g at 4uC for 10 min to pellet unbroken cells. Intact parasites and large debris were resuspended in 10 ml SMDI buffer and disrupted once more by French press at a pressure of 1000 PSI, medium setting. The pooled supernatant was centrifuged at 25,0006g at 4uC for 20 min. The supernatant was saved for analysis as the cytosolic fraction. The pellet was resuspended in 10 ml of 30% Percoll in SMDI buffer. After centrifugation at 75,0006g in an ultracentrifuge (Rotor TLA 100.3; 30,000 rpm) at 4uC for 25 min, the top band was collected from the self-generated gradient. The band was diluted in SMDI buffer and spun at 100,0006g for 90 min. at 4uC (Rotor TLA 100.3; 40,000 rpm). A band collected between the buffer and resultant Percoll cushion contained the T. gondii ghosts consisting of membranes and cytoskeleton [18]. Membrane fraction. To isolate the membrane fraction the T. gondii ghosts were resuspended in an equal volume of 2% thioglucopyranoside in 40 mM Tris pH 7.6 by pipeting the mixture up and down 10 times (suspension kept on ice) followed by a brief vortex. After centrifugation at 20,0006g in an Eppendorff centrifuge for 20 min. at 4uC, the supernatant was saved as membrane fraction (extraction 1). This extraction was repeated twice with 300 ml of 1% thioglucopyranoside (to make extractions 2 and 3). These fractions were then frozen in liquid nitrogen and stored at 280uC until used for protein analysis. Cytoskeleton fraction. Insoluble material, which remained after the membrane fraction extraction, was washed twice with 40 mM Tris pH 7.6. Following this washing step the material was solubilized in 500 ml of urea lysis buffer (7.5 M Urea, 2.5 M Thiourea, 40 mM Tris pH 7.6, 2.5% Octyl-b-glucoside, 6.25 mM TCEP, 1.256 Proteinase inhibitor) followed by homogenization on ice 10 times using a Potter homogenizer. The material was then centrifuged at 8,000 rpm for 10 min at 4uC and the supernatant was collected and saved. This extraction was repeated twice on the remaining pellet using 300 ul of the urea lysis buffer and the supernatants were pooled with the initial extraction to produce the cytoskeleton fraction. This fraction was then frozen in liquid nitrogen and stored at 280uC until used for protein analysis. High throughput mass spectrometry Nanospray LC-MS/MS was performed on a LTQ linear ion trap mass spectrometer (LTQ, Thermo, San Jose, CA) interfaced with a TriVersa NanoMate nanoelectrospray ion source (Advion BioSciences, Ithaca, NY). An Ultimate Plus nano-HPLC system with a Famous autosampler (Dionex Corporation, Sunnyvale, CA), was coupled with the TriVersa NanoMate. Peptides were Validation of Gene Predictions loaded on a C18 m-PrecolumnTM Cartridge (5 mm, 100 A˚, 300 mm i.d.9 5 mm) from the autosampler with a 25 ml sample loop at a flow rate of 15 ml/min. After injection of sample, 20 mL, and washing for 20 minutes, the precolumn was switched in line with the analytical column, a C18 PepMap100, 3 mm, 100 A˚, 75 mm i.d. 9 150 mm (Dionex Corporation, Sunnyvale, CA). Mobile phase B (80% acetonitrile/water+0.1% formic acid) was increased from 2% to 55% over 70 minutes, held for 5 minutes, increased to 95% over 20 minutes and held at 95% B for 5 minutes. The flow rate used was 250 nL/min and mobile phase A consisted of 5% acetonitrile/water+0.1% formic acid. The four most intense ions having a charge state between +2 to +4, determined from an initial survey scan from 300–1800 m/z, were selected for zoom scan and MS/MS. MS/MS was performed using an isolation width of 2 m/ z, normalized collision energy of 35%, a minimum signal intensity of 1000 counts, and the dynamic exclusion option enabled. Once a certain ion is selected twice for MS/MS in 30 sec, this ion is excluded from being selected again for MS/MS during the next period of 120 sec. Dta files were created from the raw LTQ mass spectrometer LC-MS/MS data. The created dta files were then merged using the merge script tool from Matrix Science (http:// www.matrixscience.com). The subsequent combined merge file was used to search the database of compiled T. gondii proteins using the following parameters with MASCOT [24] (in-house): trypsin, 2 missed cleavages; variable modifications of carbamido- methylation (Cys), deamidation (Asn and Gln) and oxidation (Met); monoisotopic masses; peptide mass tolerance of 3.0 Da; product ion mass tolerance of 0.6 Da. Peptides with a 95% significance score were accepted only. Each identified protein sequence was categorized according to the highest ion score of all of it’s assigned peptides; 76% of the proteins had a high ion score more than 60, while 24% of the proteins had a high ion score between 30 and 60. EST data analysis The EST dataset was obtained by downloading the NCBI EST ‘‘others’’ database (ftp://ftp.ncbi.nih.gov/blast/db/). The FASTA definition lines were searched and the sequences were selected for the organism name ‘‘Toxoplasma gondii’’. A custom T. gondii EST database was constructed for the resulting 129,736 sequences. A TBLASTN (with an e-value requirement of 10210 and without low complexity filtering) search was performed for each protein sequence against the custom database of T. gondii EST sequences. For each query sequence, the EST alignments were filtered to include only the ESTs with a sequence identity of greater or equal to 90% and an alignment length of 30 or more residues. Supporting Information Table S1 Sequence annotation (PFAM, Transmembrane do- mains, Signal peptides) for sequence of each prediction type. Found at: doi:10.1371/journal.pone.0003899.s001 (0.04 MB DOC) Acknowledgments We thank ToxoDB.org for making Toxoplasma related experimental and computational data available. Author Contributions Conceived and designed the experiments: RHA LW AF. Performed the experiments: FYC EN LW. Analyzed the data: JMD CJMA EN DR RHA KK AF. Wrote the paper: JMD AF. PLoS ONE | www.plosone.org 11 December 2008 | Volume 3 | Issue 12 | e3899