Tissue culture and parasite purification
Vero cell cultures were maintained in RPMI-1640 medium (Sigma) supplemented with 5% fetal calf serum (Cultilab), 2.05 mM glutamine, 50 U penicillin/streptomycin in T-25 or 75 tissue culture flasks at 37°C and 5% CO2. The cultures were trypsinized at least once a week. N. caninum tachyzoites from the Nc-1 isolate were maintained in Vero cell monolayers and purified by exclusion chromatography in Sephadex G-25 (PD-10 columns; GE).
The N. caninum data from high-resolution mass spectrometry-based proteomics was obtained as previously described. Proteins present in the ESA and in the discharged tachyzoites (parasites submitted to an ethanol secretion stimulus) were identified using shotgun LC-MS/MS. The protein IDs were generated after separation of the N. caninum proteins by strong cation exchange chromatography. From this work, 615 proteins were identified in ESA and 2,011 proteins quantified in the discharged tachyzoites; however, few properties about MIC2 proteins family were deeply analysed. Therefore, in this article, all the PSMs were analyzed in detail for NcMIC2-like 1 and NcMIC2 identification/quantification.
The N. caninum tachyzoite protein extract, prepared as described previously, was dissolved (50 μg) in a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% carrier ampholyte mixture (pH 3–5.6 NL in Immobiline Drystrip gels; GE), 20 mM dithiothreitol and supplemented with protease inhibitors (1/100, Protease Inhibitor cocktail; Sigma-Aldrich). The samples were loaded on 7 cm IPG strips (pH 3–5.6 NL), rehydrated and focused in an automated overnight run (IPGPhor) using 10–14 h of rehydration followed by a step voltage focusing procedure (20 min 100 V, 30 min 300 V, 1.2 h 1,000 V, 15 min 5,000 V, followed by 5,000 V until a total of 5 kVh was reached). After focusing, the strips were incubated with 30 mM dithiothreitol and 135 mM iodoacetamide in the equilibration buffer (50 mM Tris, 6 M urea, 2% SDS, 30% glycerol, pH 8.8) for 15 min. The proteins were separated by 10% SDS-PAGE and the gels stained with Coomassie Brilliant Blue G 250. Gel images were acquired with LabScan v5.0 software on a flatbed scanner (Image Scanner; GE).
The 2D gels of N. caninum tachyzoite protein extracts were transferred to a PVDF membrane (Immobilon-P; Millipore) with a semi-dry system (TE 77 PWR; Amersham Biosciences) using transfer buffer (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20% methanol) at 0.81 mA/cm2 for 1.15 h. The blot was blocked with 0.8% porcine gelatin (Sigma-Aldrich) diluted in phosphate-buffered saline containing 0.05% Tween (PBST) for 1 h at 37°C. The antiserum anti-NcMIC2-like1 was diluted (1/2,000) in 0.2% porcine gelatin PBST and incubated overnight at 4°C. After washing (three times with PBST), the blot was incubated with anti-rabbit-immunoglobulin conjugated to HRP (1/10,000; ZyMed-Invitrogen) for 1 h at 37°C and then washed three times with PBST. The signal was obtained with an enhanced chemiluminescent substrate for HRP detection (SuperSignal West Pico Chemiluminescent Substrate; Pierce). The blots were scanned using the Image Scanner.
The spots were washed once with milliQ water and twice with 50 mM ammonium bicarbonate (pH 8.0) followed by shrinkage with 100% acetonitrile (ACN). For protein digestion, the spot was incubated overnight with trypsin (Promega) at 37°C. The peptides were extracted with 100% ACN, dried in a SpeedVac vacuum concentrator, and analyzed by RP-nano-LC-MS/MS. After resuspension in 10% formic acid, the peptides were loaded into a LTQ Orbitrap XL ETD mass spectrometer (Thermo Electron, Bremen, Germany) coupled to an Agilent 1200 HPLC system (Agilent Technologies), using a 20×100 mm trap column (Aquat C18, 5 mm; Phenomenex, Torrance, CA) and a 40 cm × 50 mm analytical column (ReproSil-Pur C18-AQ, 3 mm; Dr Maisch GmbH, Ammerbuch, Germany). A 60 min run was carried out using elution gradients with solvent A (0.6% acetic acid) and with solvent B (0.6% acetic acid in 80% ACN) with a flow rate of 100 nL/min. The five most intense peptide ions were selected for fragmentation by collision-induced dissociation. The outputs were compared with data in the N. caninum predicted protein database (ToxoDB 28) using the Mascot software version 2.4.01 (Matrix Science).
Carbamidomethylation of cysteine and oxidation of methionine were set as fixed and variable modifications, respectively. The database search was performed with a peptide tolerance of 50 ppm and product ion tolerance of 0.6 Da, allowing two missed cleavages.