DOI: 10.5061/dryad.tht76hdzp
title: Comparative Proteomics Reveals Characteristic Proteins on Praziquantel-resistance in Schistosoma mansoni

description: 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.
description: <strong>Preparation of protein extracts:
 </strong>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.
 <strong>Two-dimensional electrophoresis:</strong>
 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.<br> 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.<br> 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. <strong>In-gel digestion and
 peptide preparation for mass spectrometry analysis: </strong>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.<br>   <strong>Nanoflow LC-MS/MS
 analysis and protein identification:</strong><i> </i>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.
 <strong>Bioinformatics: </strong>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 &lt;
 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”.
description: Usage information included in README included with data
 files.