DOI: 10.25345/c5g73777t
title: MassIVE MSV000089877 - An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii
title: Protein samples were analyzed by Multidimensional Protein Identification Technology (MudPIT), as described previously. Briefly, precipitated proteins were resuspended in 30uL of 100mM Tris pH 8.5 with 8M urea to denature proteins. Cysteines were reduced and alkylated prior to digestion with recombinant LysC and modified trypsin. Reactions were quenched by the addition of formic acid to the final concentration of 5%. After digestion, peptide samples were pressure-loaded onto 100 um fused silica microcapillary columns packed first with 9 cm of reverse phase material, followed by 3 cm of 5um Strong Cation Exchange material, followed by 1 cm of 5um C18 RP. The loaded microcapillary columns were placed in-line with a 1260 Quartenary HPLC. The application of a 2.5 kV distal voltage electrosprayed the eluting peptides directly into orbitrap Elite mass spectrometers equipped with a custom-made nano-LC electrospray ionization source. Full MS spectra were recorded on the eluting peptides over a 400 to 1600 m/z range, followed by fragmentation in the ion trap on the first to fifth most intense ions selected from the full MS spectrum. Dynamic exclusion was enabled for 90 s. Mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur data system. RAW files were extracted into .ms2 file format using RawDistiller v. 1.0, in-house developed software. RawDistiller D(g, 6) settings were used to abstract MS1 scan profiles by Gaussian fitting and to implement dynamic offline lock mass using six background polydimethylcyclosiloxane ions as internal calibrants. MS/MS spectra were first searched using ProLuCID with a mass tolerance of 10 ppm for peptide and fragment ions. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database containing 90240 human proteins (NCBI 2021-11-23 release) and 8311 Toxoplasma gondii proteins (NCBI 2021-11-05) , as well as 426 common contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database, for a total search space of 180482 amino acid sequences. Masses of 57.0215 Da was differentially added to cysteine residues to account for alkylation by CAM and 15.9949 Da were differentially added to methionine residues. DTASelect v.1.9 was used to select and sort peptide/spectrum matches (PSMs) passing the following criteria set: PSMs were only retained if they had a DeltCn of at least 0.08; minimum XCorr values of 1.8 for singly-, 2.1 for doubly-, and 2.5 for triply-charged spectra; peptides had to be at least 7 amino acids long. Results from each sample were merged and compared using CONTRAST. Combining all replicate runs, proteins had to be detected by at least 2 peptides and/or 2 spectral counts. Proteins that were subsets of others were removed using the parsimony option in DTASelect on the proteins detected after merging all runs. Proteins that were identified by the same set of peptides (including at least one peptide unique to such protein group to distinguish between isoforms) were grouped together, and one accession number was arbitrarily considered as representative of each protein group. NSAF7 was used to create the final reports on all detected peptides and non-redundant proteins identified across the different run.