The extensive use of Praziquantel (PZQ), the only drug available to treat schistosomiasis, has brought concern about the emergence of PZQ-resistance/tolerance by Schistosoma spp., thus reaffirming an urge for the development of new treatment alternatives. Therefore, it is imperative and urgent to study this phenomenon trying to understand what is involved in its occurrence. Studies of Schistosoma spp. genome, transcriptome and proteome are crucial to better understand this situation. By stepwise drug pressure from a fully susceptible parasite strain, our group selected a S. mansoni variant strain stably resistant to PZQ and isogenic to its fully susceptible parental counterpart, except for the genetic determinants of PZQ-resistance phenotype. Based on this, the objective of this study was to compare the proteomes of both strains, identifying proteins from male and female adult worms of PZQ-resistant and PZQ-susceptible strains, exposed and not exposed to PZQ, which were separated by high-resolution two-dimensional electrophoresis and sequenced by high throughput LC-MS/MS. Likewise, this work is extremely relevant since for the first time the proteome of a S. mansoni PZQ-resistant strain is studied and compared to the proteome of the respective S. mansoni PZQ-susceptible strain. This study identified 60 S. mansoni proteins, some of which differentially expressed in either strain, which may putatively be involved in the PZQ-resistance phenomenon. This information represents substantial progress towards deciphering the worm proteome. Furthermore, these data may constitute an informative source for further investigations into PZQ-resistance and increase the possibility of identifying proteins related to this condition, possibly contributing to avoid or decrease the likelihood of development and spread of PZQ-resistance. This is an innovative study that opens doors to PZQ-resistance surveys, contributing to discover a solution to PZQ-resistance problem, as suggests new potential targets for study.

Preparation of protein extracts: Schistosoma mansoni adult parasite protein extracts were obtained using Trizol protocol, according to the manufacturer’s instructions. Briefly, the parasites were lysed and homogenized directly in Trizol reagent at room temperature (≈ 25 ºC). The homogenized samples were incubated at room temperature to permit complete dissociation of the nucleoprotein complex. After homogenization, we proceeded to separation phase, adding chloroform and centrifugation of samples. The aqueous phase was removed and the interphase and organic phenol-chloroform phase was used for protein isolation procedure. Next, isopropyl alcohol precipitation was performed and the pellet was solubilized in SBI buffer (7 mol/L urea, 2 mol/L thiourea, 0.015 mol/L DHPC, 0.5% triton X-100, 0.02 mol/L DTT and complete mini protease inhibitor cocktail tablets), according to and stored at  ̶ 80 ºC until use. Protein concentration in protein extracts was measured by Bradford assay, and the quality of the extract was verified in 12% uniform SDS-PAGE gels.

Two-dimensional electrophoresis: Each experiment with two-dimensional electrophoresis (2-DE) gels was performed in triplicate with 240 μg of protein extracts, for each group. To prepare samples for 2-DE, protein samples were diluted in rehydration solution containing 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 0.5% IPG buffer, 1% DTT, and 0.002% bromophenol blue. The rehydration was carried out passively overnight during 12 h in a 13 cm pH 3-10 strip. The strips were then applied on an Ettan IPGphor 3 System (GE Healthcare, Piscataway, NJ, USA), for protein separation by isoelectric focusing (IEF), using the following conditions developed for this work: at a constant current 50 μA/strip, the voltage program started with a gradient up to 3.5 kV in 3 h, then a step of 3 h at 3.5 kV, then a total voltage of 64.0 kVh to the end.
After focusing, the strips containing proteins were reduced in an equilibration solution (50 mol/L Tris-HCl, pH 8.8, 6 mol/L urea, 20% glycerol, 2% SDS) containing 2% DTT, and then alkylated in the same solution containing 2.5% IAA. The Immobilized pH gradient (IPG) strips and molecular weight standards were then transferred to the top of 12% uniform SDS-PAGE gels, and sealed with 0.5% agarose. The second dimension was carried out using a Protein Plus Dodeca cell system (Bio-Rad Laboratories, Inc., Hercules, CA) under an initial current of 15 mA/gel for 15 min, followed by increasing the current to 50 mA/gel until the end of the run.
For 2-DE experiments, at least three replicas of two-dimensional polyacrylamide gel electrophoresis were performed for each group, confirming the reproducibility of the experimental procedure. Gels were fixed in 40% methanol/10% acetic acid solution and stained with coomassie brilliant blue R-350. The spots were normalized and evaluated using the ImageMaster® 2D Platinum 7.0 software (GE Healthcare Bio-Sciences, Uppsala, Sweden), according the number of spots and matching, and the results were exported and evaluated in SIMCA-P software (Umetrics, Umeå, Sweden). Here, we built PCA models, from which it was possible to obtain statistically valid spots between groups using t-test and Jack knife.

In-gel digestion and peptide preparation for mass spectrometry analysis: The selected protein spots from two biological samples for each group were manually excised, distained, reduced, alkylated, and digested in gel with trypsin from the corresponding 2-DE gel for mass spectrometry (MS) identification. First, spots were washed in ultrapure water, and then distained in a solution containing 50% methanol/2.5% acetic acid, for 2 h at room temperature. This step was repeated until clear of blue stain. The gel fragments were incubated in 100% ACN with occasional vortexing, until gel pieces became white and shrank. Then, the solution was removed and spots were completely dried, and ready for digestion. The in-gel digestion with trypsin modified sequencing grade reagents was done according to. Briefly, protein digestion was conducted at 37 ºC overnight. After the incubation, the supernatant was transferred to a clean tube, and 30 μL of 5% FA/60% ACN solution was added to gel spots for the extraction of tryptic peptides. This procedure was performed 2 times during 30 min, under constant agitation. The supernatant was pooled to the respective tube containing the initial peptide solution. This solution was dried in a speedvac concentrator Thermo Fisher Scientific (Waltham, MA) and the peptides were re-suspended in 8 μL of 0.1% FA solution. The peptides were desalted in reverse phase micro-columns ZipTip® C18 (Millipore Corporation, Billerica, MA), according to manufacturer’s instructions. Peptides were dried again and resuspended in 50% ACN/0.1% TFA solution.
 

Nanoflow LC-MS/MS analysis and protein identification: The digested peptides (50 µg) were analyzed in an Easy-nLC II nanoflow liquid chromatograph system (nano-LC-MS/MS) (Thermo Fisher Scientific, Waltham, MA) in tandem with a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific), which was equipped with a nano electrospray Nano-Flex II (Thermo Fisher Scientific) operating in a positive ion mode. Nano electrospray voltage was set to 4.5 kV and source temperature at 220 °C. The precursor ion was isolated using the data-dependent acquisition mode with a 2 m/z isolation width and sequentially the ten most intense ions were selected for fragmentation event by collision induced dissociation (CID), at 35% normalized collision energy and 10 ms activation time. Maximum ion injection times were set to 100 ms for MS and 500 ms for MS/MS, with a resolution of 60,000 and a scan within m/z range from 400 to 2000. Chromatographic separation was carried out a Thermo C18 capillary column (10 cm × 75 µm, 3 µm, 120 Å) protected by a guard Thermo C18 capillary column (2 cm × 100 µm i.d., 5 µm particle, 120 Å pore size). Ultrapure water containing 0.1% FA (solvent A) and ACN containing 0.1% FA (solvent B) was used as mobile phase. The separation was performed at room temperature with a constant flow rate of 0.3 µL/min, with a total acquisition time of 90 min., by employing the elution program as follows: linear gradient of 5% of solvent B during 5 min, ranging to 80% of solvent B over 85 min. Data acquisition was controlled by Xcalibur® 2.0.7 Software (Thermo Fisher Scientific) and converted in mgf files using MassMatrix® MS Data file conversion version 3.9 software.

Bioinformatics: The list of peptide and fragment mass values generated by the mass spectrometer for each spot were submitted to a MS/MS ion search using the Mascot® 2.0 software online search engine (Matrix Science Inc, Boston, MA, USA) to search the Nanoflow LC-MS/MS data against the NCBInr database Schistosoma_mansoni_NCBI_112014, November 2014. Mascot® software was set with two tryptic missed cleavages, a peptide ion mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.02 Da, a peptide charge state of 2+ and 3+, a variable modifications of methionine (oxidation), and a fixed modifications of cysteine (carbamidomethylation). During the analysis, our samples were checked against a contaminant database supplied by the Global Proteome Machine. All validated proteins had at least two independent spectra, with greater than 95.0% probability estimated by the Peptide Prophet algorithm of being present in the S. mansoni database as at least one unique peptide. To avoid random matches, only ions with individual score above of the indicated by the Mascot® to identity or extensive homology (p < 0.05) were considered for protein identification. However, when Mascot® score was not significant, but the percentage coverage and root mean squared error (RMSE) were in the same range as those of proteins with a significant match, proteins were deemed identified if additional parameters, such as its calculated pI and Mw, were in agreement with those observed for the actual gel spot and the species matched was S. mansoni. The molecular function and biological process were assigned for the proteins identified according to information obtained from the Gene Ontology® (GO) database. The exact annotation for each protein was used in most cases. However, the catalytic activity category was used for all proteins with molecular function associated with (GTPase, hydrolase, isomerase, kinase, ligase, lyase, oxidoreductase, transcription, and transferase activities). Binding category was used for all types of ligand identified (actin, ATP, calcium, GTP, magnesium ion, metal ion, protein domain specific, and nucleotide bindings). Furthermore, there was other molecular function categories classified such as chaperone, motor, regulation of muscle contraction, structural, and transport. The proteins that had no associated known function were classified as “unknown”.

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