Secreted Proteomes of Different Developmental Stages of the Gastrointestinal Nematode Nippostrongylus brasiliensis
2014; Elsevier BV; Volume: 13; Issue: 10 Linguagem: Inglês
10.1074/mcp.m114.038950
ISSN1535-9484
AutoresJavier Sotillo, Alejandro Sánchez‐Flores, Cinzia Cantacessi, Yvonne Harcus, Darren Pickering, Tiffany Bouchery, Mali Camberis, Shiau-Choot Tang, Paul Giacomin, Jason Mulvenna, Makedonka Mitreva, Matthew Berriman, Graham Le Gros, Rick M. Maizels, Alex Loukas,
Tópico(s)Parasites and Host Interactions
ResumoHookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yielding∼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS—52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins—astacin metalloproteases and CAP-domain containing SCP/TAPS—were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model. Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yielding∼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS—52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins—astacin metalloproteases and CAP-domain containing SCP/TAPS—were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model. Nematodes belonging to the order Strongylida are, from an epidemiological and a socio-economic perspective, among the most relevant parasites in the world. Within this suborder, species from the genera Necator and Ancylostoma (also known as hookworms) infect more than 700 million people in tropical areas, and are considered to cause one of the most important human helminth infections along with schistosomiasis in terms of disability-adjusted life years lost (1.Hotez P.J. Brooker S. Bethony J.M. Bottazzi M.E. Loukas A. Xiao S. Hookworm infection.N. Engl. J. 2004; 351: 799-807Crossref PubMed Scopus (489) Google Scholar, 2.Hotez P.J. Fenwick A. Savioli L. Molyneux D.H. 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L3 penetrate the skin of the host and migrate through the subcutaneous connective tissue where they enter the circulatory system and travel to the lungs before exiting into the alveolar spaces and moulting to the L4 stage. From here, they migrate up the trachea and are swallowed, finally entering the gastrointestinal tract as L4 larvae and maturing to sexually dioecious male and female adults in the small intestine where they feed and mate. The N. brasiliensis-rodent model has been widely used as a model for human hookworm disease (4.Camberis M. Le Gros G. Urban Jr., J. Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus.Current protocols in immunology. 2003; (edited by John E. Coligan. [et al.] Chapter 19, Unit 19 12)Crossref PubMed Google Scholar, 7.McSorley H.J. Loukas A. The immunology of human hookworm infections.Parasite Immunol. 2010; 32: 549-559PubMed Google Scholar, 8.McSorley H.J. Maizels R.M. Helminth infections and host immune regulation.Clin. 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Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity.Nature. 2010; 464: 1367-1370Crossref PubMed Scopus (1619) Google Scholar, 16.Shin E.H. Osada Y. Chai J.Y. Matsumoto N. Takatsu K. Kojima S. Protective roles of eosinophils in Nippostrongylus brasiliensis infection.Int. Arch. Allergy Immunol. 1997; 114: 45-50Crossref PubMed Scopus (41) Google Scholar). Despite an abundance of studies addressing the mechanistic aspects of rodent immunity to N. brasiliensis infections, there is a distinct paucity of molecular information about the parasite itself. A search of the NCBI database for N. brasiliensis retrieves only 116 proteins (most of them redundant), although an early transcriptomic analysis (pre-Next Generation Sequencing technologies) described ∼1300 expressed sequence tags corresponding to 742 distinct genes (17.Harcus Y.M. Parkinson J. Fernandez C. Daub J. Selkirk M.E. Blaxter M.L. Maizels R.M. Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites.Genome Biol. 2004; 5: R39Crossref PubMed Google Scholar). Herein we present the first high-throughput proteomic characterization of the proteins present in the excretory/secretory products (ESP) 1The abbreviations used are:ESPExcretory/secretory productsBLASTBasic Local Alignment Search ToolCAPCysteine-rich proteinAntigen 5Pathogenesis-related protein 1 domainDTEDithioerythritolemPAIexponential modified protein abundance indexGOGene OntologyIAMIodoacetamideNExwhole worm extractOGEOFF-GEL electrophoresisSCPSperm coat proteinTAPSTpx-1Antigen 5Pathogenesis-related protein 1, SCP-likeVALVenome allergen/Ancylostoma secreted protein-Like. 1The abbreviations used are:ESPExcretory/secretory productsBLASTBasic Local Alignment Search ToolCAPCysteine-rich proteinAntigen 5Pathogenesis-related protein 1 domainDTEDithioerythritolemPAIexponential modified protein abundance indexGOGene OntologyIAMIodoacetamideNExwhole worm extractOGEOFF-GEL electrophoresisSCPSperm coat proteinTAPSTpx-1Antigen 5Pathogenesis-related protein 1, SCP-likeVALVenome allergen/Ancylostoma secreted protein-Like. of N. brasiliensis infective stage L3 and intestinal-dwelling adult worms based on a full exploration of the transcriptome using Illumina-based sequencing technology. Large-scale data comparisons between the secreted proteome from N. brasiliensis and available genomic and proteomic data for N. americanus were performed (18.Tang Y.T. Gao X. Rosa B.A. Abubucker S. Hallsworth-Pepin K. Martin J. Tyagi R. Heizer E. Zhang X. Bhonagiri-Palsikar V. Minx P. Warren W.C. Wang Q. Zhan B. Hotez P.J. Sternberg P.W. Dougall A. Gaze S.T. Mulvenna J. Sotillo J. Ranganathan S. Rabelo E.M. Wilson R.K. Felgner P.L. Bethony J. Hawdon J.M. Gasser R.B. Loukas A. Mitreva M. Genome of the human hookworm Necator americanus.Nat. Genet. 2014; (doi: 10.1038/ng.2875)Crossref Scopus (139) Google Scholar). This comprehensive analysis of the proteins and mRNAs produced by N. brasiliensis provides new insights into the molecular interactions at the host-parasite interface and highlight the molecular similarities between N. brasiliensis and N. americanus, emphasizing the utility of this model rodent nematode for exploring the immunobiology of hookworm infections, and as a model for the discovery and development of new therapeutic approaches to controlling gastrointestinal nematodes. Excretory/secretory products Basic Local Alignment Search Tool Cysteine-rich protein Pathogenesis-related protein 1 domain Dithioerythritol exponential modified protein abundance index Gene Ontology Iodoacetamide whole worm extract OFF-GEL electrophoresis Sperm coat protein Tpx-1 Pathogenesis-related protein 1, SCP-like Venome allergen/Ancylostoma secreted protein-Like. Excretory/secretory products Basic Local Alignment Search Tool Cysteine-rich protein Pathogenesis-related protein 1 domain Dithioerythritol exponential modified protein abundance index Gene Ontology Iodoacetamide whole worm extract OFF-GEL electrophoresis Sperm coat protein Tpx-1 Pathogenesis-related protein 1, SCP-like Venome allergen/Ancylostoma secreted protein-Like. N. brasiliensis was maintained in Sprague-Dawley rats as previously described (4.Camberis M. Le Gros G. Urban Jr., J. Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus.Current protocols in immunology. 2003; (edited by John E. Coligan. [et al.] Chapter 19, Unit 19 12)Crossref PubMed Google Scholar, 19.Lawrence R.A. Gray C.A. Osborne J. Maizels R.M. Nippostrongylus brasiliensis: cytokine responses and nematode expulsion in normal and IL-4-deficient mice.Exp. Parasitol. 1996; 84: 65-73Crossref PubMed Scopus (84) Google Scholar) and in accordance with UK Home Office and local Ethical Review Committee approvals. Infective L3s were prepared from two-week rat fecal cultures with careful preparation to ensure 100% viability. Adult worms were recovered from gastrointestinal tissue using a Baermann apparatus on day 6 post-infection following subcutaneous injection of 3000 infective L3. In addition, eggs and lung-stage larvae were included in the transcriptomic analysis to ensure that transcripts encoding proteins present in the subsequent L3 and adult worm secretomes were fully represented. The RNA extraction was performed as described previously by Harcus et al. (17.Harcus Y.M. Parkinson J. Fernandez C. Daub J. Selkirk M.E. Blaxter M.L. Maizels R.M. Signal sequence analysis of expressed sequence tags from the nematode Nippostrongylus brasiliensis and the evolution of secreted proteins in parasites.Genome Biol. 2004; 5: R39Crossref PubMed Google Scholar). Briefly, total RNA was extracted from different stages of N. brasiliensis (egg, L3, L4, and adult) and homogenized in 1 ml Trizol (Invitrogen, Carlsbad, CA). The homogenate was centrifuged (12,000 × g, 10 min), and the supernatant extracted with chloroform before isopropanol precipitation of RNA from the aqueous phase and DNase treatment. Polyadenylated (Poly(A)+) RNA was purified from 10 μg of total RNA, fragmented to a length of 100–500 bases, reverse-transcribed to cDNA, adaptor-ligated and paired-end sequenced on a Genome Analyzer II (Illumina, San Diego), obtaining paired-end reads with a length of 108 bp each. The resulting data passed a quality control (QC) where sequencing adapters and reads with low quality (< 30 PHRED) were removed. Transcript reconstruction was performed using the Trinity assembler (rev 2013–02-25) (20.Grabherr M.G. Haas B.J. Yassour M. Levin J.Z. Thompson D.A. Amit I. Adiconis X. Fan L. Raychowdhury R. Zeng Q. Chen Z. Mauceli E. Hacohen N. Gnirke A. Rhind N. di Palma F. Birren B.W. Nusbaum C. Lindblad-Toh K. Friedman N. Regev A. Full-length transcriptome assembly from RNA-Seq data without a reference genome.Nat. Biotechnol. 2011; 29: 644-652Crossref PubMed Scopus (12682) Google Scholar) using default parameters, from a pool of reads for all life stages. Also, downstream analysis for transcript abundance estimation and differential expression between life stages was performed following the protocol described in (21.Haas B.J. Papanicolaou A. Yassour M. Grabherr M. Blood P.D. Bowden J. Couger M.B. Eccles D. Li B. Lieber M. Macmanes M.D. Ott M. Orvis J. Pochet N. Strozzi F. Weeks N. Westerman R. William T. Dewey C.N. Henschel R. Leduc R.D. Friedman N. Regev A. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis.Nat. Protoc. 2013; 8: 1494-1512Crossref PubMed Scopus (4812) Google Scholar). Reconstructed transcripts were annotated using the Trinotate pipeline (http://trinotate.sourceforge.net/), a blastx search against the NEMBASE4 (http://www.nematodes.org/nembase4/), and against the entire genome sequence of the human hookworm N. americanus (18.Tang Y.T. Gao X. Rosa B.A. Abubucker S. Hallsworth-Pepin K. Martin J. Tyagi R. Heizer E. Zhang X. Bhonagiri-Palsikar V. Minx P. Warren W.C. Wang Q. Zhan B. Hotez P.J. Sternberg P.W. Dougall A. Gaze S.T. Mulvenna J. Sotillo J. Ranganathan S. Rabelo E.M. Wilson R.K. Felgner P.L. Bethony J. Hawdon J.M. Gasser R.B. Loukas A. Mitreva M. Genome of the human hookworm Necator americanus.Nat. Genet. 2014; (doi: 10.1038/ng.2875)Crossref Scopus (139) Google Scholar) to confirm their origin and rule out contamination from host or bacterial sources. Proteins were then conceptually translated from the predicted coding domains of individual cDNA sequences. Adult worms and L3 were recovered in saline at 37 °C, washed 6× in saline and 6× in RPMI 1640 containing 100 μg/ml penicillin and 100 U/ml streptomycin (complete medium), and viability was determined by microscopy. The collection of ESP from L3 larvae and adult worms in vitro followed previously described protocols (6.Holland M.J. Harcus Y.M. Riches P.L. Maizels R.M. Proteins secreted by the parasitic nematode Nippostrongylus brasiliensis act as adjuvants for Th2 responses.Eur. J. Immunol. 2000; 30: 1977-1987Crossref PubMed Scopus (118) Google Scholar, 22.Healer J. Ashall F. Maizels R.M. Characterization of proteolytic enzymes from larval and adult Nippostrongylus brasiliensis.Parasitology. 1991; 103: 305-314Crossref PubMed Scopus (35) Google Scholar). Briefly, parasites recovered as above were cultured in complete medium supplemented with glucose (to 1%), at 37 °C in an atmosphere of 5% CO2. Supernatants were collected daily from worms cultured for 2–7 days, pooled, centrifuged at 400 × g for 5 min to remove any eggs, passed through a 0.2 μm Millex filter, concentrated over a 10,000 Da Amicon membrane, and stored at ∼1.0 mg/ml in PBS at −80 °C until required. N. brasiliensis adult worm somatic extract (NEx) was prepared by homogenization on ice of freshly-isolated adult worms in PBS followed by centrifugation at 12,000 × g for 30 min and recovery of the supernatant. The ESP of N. brasiliensis was subjected to two-dimensional gel electrophoresis (2DE) and silver stained as described previously (23.Hewitson J.P. Harcus Y.M. Curwen R.S. Dowle A.A. Atmadja A.K. Ashton P.D. Wilson A. Maizels R.M. The secretome of the filarial parasite, Brugia malayi: proteomic profile of adult excretory-secretory products.Mol. Biochem. Parasitol. 2008; 160: 8-21Crossref PubMed Scopus (213) Google Scholar). Briefly, 10 μg of protein was resuspended in 125 μl of rehydration buffer (7 m Urea (Electran, BDH, Hinckley UK), 2 m thiourea (BDH), 4% CHAPS (Sigma, St. Louis, Mo.), 65 mm DTE (Sigma), 0.8% IPG buffer 3–10 (GE Healthcare, Little Chalfont UK), and trace bromphenol blue (Sigma)), and used to rehydrate a 7 cm strip pH 3–10 (Immobiline; GE Healthcare) for 14 h at 20 °C. Proteins were then subjected to isoelectric focusing on an IPGphor (Pharmacia Biotech, Sandwich UK) at 20 °C using the following program: 1) 500 V for 30 min; 2) 1000 V for 30 min; 3) gradient to 8000 V for 6 h; 4) total ∼20 kVh. Strips were reduced and alkylated as described previously (24.Sotillo J. Valero L. Sanchez Del Pino M.M. Fried B. Esteban J.G. Marcilla A. Toledo R. Identification of antigenic proteins from Echinostoma caproni (Trematoda) recognized by mouse immunoglobulins M, A, and G using an immunoproteomic approach.Parasite Immunol. 2008; 30: 271-279Crossref PubMed Scopus (41) Google Scholar), and proteins were separated by molecular weight using NuPAGE 4–12% Bis-Tris ZOOM gels (Invitrogen) and Nu-PAGE MES SDS running buffer (Invitrogen) for 2 h 10 min at constant 100 V. Gels were silver stained using PlusOne (GE Healthcare) with a few modifications as described by Yan et al. (25.Yan J.X. Wait R. Berkelman T. Harry R.A. Westbrook J.A. Wheeler C.H. Dunn M.J. A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry.Electrophoresis. 2000; 21: 3666-3672Crossref PubMed Scopus (647) Google Scholar). In-gel digestion of the 56 spots sliced from the gel was performed as described by Mulvenna et al. (26.Mulvenna J. Hamilton B. Nagaraj S.H. Smyth D. Loukas A. Gorman J.J. Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, Ancylostoma caninum.Mol. Cell Proteomics. 2009; 8: 109-121Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) with some modifications. Gel spots were washed in 50% acetonitrile, 25 mm NH4CO3 for 5 min three times at 37 °C, and then dried under a vacuum centrifuge. The spots were then reduced in 20 mm dithiothreitol (DTT) for 1 h at 65 °C, the supernatant was removed, and samples were alkylated by the addition of 55 mm iodoacetamide (IAM) and incubated at room temperature in darkness for 40 min. Gel spots were washed 3× in 25 mm NH4CO3 and dried in a vacuum centrifuge. The dried spots were rehydrated with 20 μl of 40 mm NH4CO3 containing 20 μg/ml trypsin (Sigma) for 45 min at 22 °C. An additional 50 μl of 40 mm NH4CO3, 9% acetonitrile was added to the samples and incubated overnight at 37 °C. The digest supernatant was removed from the spots, and residual peptides were removed from the gel slices by washing 3 × 0.1% TFA for 45 min at 37 °C. The original supernatant and extracts were combined and dried in a vacuum centrifuge. Samples were desalted and concentrated using Zip-Tip® (Merck Millipore, Waterford, UK) and eluted in ∼5 μl of 50% acetonitrile 0.1% TFA before mass spectral analysis. The adult and L3 ESP from N. brasiliensis (5 μg) were labeled with CyDye DIGE Fluor (minimal dyes) Cy3 (green) and Cy5 (red) respectively according to the manufacturer's instructions. Samples were combined and subjected to IEF using IPG strips (7 cm, pH 3–10 - source) under the following settings: rehydration for 14 h at room temperature, 300 V for 30 min, 1000 V for 30 min, and gradient to 5000 V for 2 h, and followed by a second dimension separation in a NuPAGE Novex 4–12% Bis-Tris ZOOM protein gel. The gels were scanned using Fujifilm Image reader FLA-5000 series V1.0. For peptide separation, OFFGEL fractionation was performed as described by Cantacessi et al. (27.Cantacessi C. Mulvenna J. Young N.D. Kasny M. Horak P. Aziz A. Hofmann A. Loukas A. Gasser R.B. A deep exploration of the transcriptome and "excretory/secretory" proteome of adult Fascioloides magna.Mol. Cell Proteomics. 2012; 11: 1340-1353Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) with some modifications. A total of 100 μg of ESP and NEx products were resuspended in 2% SDS, 20 mm DTT and were incubated at 65 °C for 1 h. Alkylation was then achieved by adding IAM to 55 mm and incubating the solution for 40 min in darkness at 22 °C. The sample was co-precipitated with 1 μg of trypsin (Sigma) and the addition of 9 volumes of methanol and was incubated overnight at −20 °C. The sample was centrifuged and the pellet was resuspended in 100 μl of 50 mm NH4CO3 and incubated for 2 h at 37 °C. After the addition of an extra 1 μg of trypsin, the sample was incubated overnight at 37 °C. The samples were then fractionated using a 3100 OFFGEL fractionator and OFFGEL kit (pH 3–10; 24-well format) (Agilent Technologies, Santa Clara, CA) according to the manufacturer's protocols. The digested proteins were diluted in peptide-focusing buffer to a final volume of 3.6 ml and 150 μl of sample were loaded into each of the 24 wells. The sample was focused in a current of 50 μA until 50 kilovolt hours (kVh) was reached. Peptide fractions were collected, dried under a vacuum centrifuge, and resuspended in 10 μl of 0.1% TFA. Finally, samples were desalted using Zip-Tip® (Merck Millipore) and dried again under a vacuum centrifuge. An AB SCIEX TOF/TOF 5800 mass spectrometer (Applied Biosystems, Foster City, CA) was used for acquiring MALDI-MS/MS data from gel spots, and samples without a positive identification were analyzed by LC-MS/MS. ESP separated by OFFGEL were analyzed by LC-MS/MS on a Shimadzu Prominance Nano HPLC coupled to an AB SCIEX Triple Tof 5600 mass spectrometer (Applied Biosystems) equipped with a nano electrospray ion source. Six microliters of sample was injected onto a 50 mm 300 μm C18 trap column (Agilent Technologies). The samples were desalted on the trap column for 5 min using 0.1% formic acid (aq) at 30 μl/min. Peptides were then eluted onto an analytical nano HPLC column (150 mm x 75 μm 300SBC18, 3.5 μm, Agilent Technologies) at a flow rate of 300 nL/min and separated using a 35 min gradient of 1–40% buffer B followed by a steeper gradient from 40–80% buffer B in 5 min. Buffer B contained 90/10 acetonitrile/0.1% formic acid, and buffer A consisted of 0.1% formic acid (aq). The mass spectrometer acquired 500 ms full scan TOF-MS data followed by 50ms full scan product ion data in an Information Dependent Acquisition, IDA, mode. Full scan TOFMS data was acquired over the mass range 350–1400, and for product ion ms/ms 80–1400 m/z ions observed in the TOF-MS scan exceeding a threshold of 100 counts and a charge state of +2 to +5 were set to trigger the acquisition of product ion, ms/ms spectra of the resultant 20 most intense ions. The data was acquired and processed using Analyst TF 1.6.1 software (ABSCIEX, Canada). Database searches were performed against NCBInr database (March 2013 version) to detect possible contamination and on the peptide sequences predicted from the N. brasiliensis transcriptomic data (20,136 entries) using MASCOT search engine v4.0 (Matrix-Science, Boston MA). The parameters used for MALDI-TOF/TOF were: enzyme; trypsin; precursor ion mass tolerance = ±0.8 Da; fixed modifications = methionine oxidation; variable modifications = carbamidomethylation; number of missed cleavages allowed = 2; charges states = +1. The parameters used for LC-TOF/TOF were similar except for the precursor ion mass tolerance = ±0.1 Da and for the charges states = +2, +3. The results from the Mascot searches were validated using the program Scaffold v.4.2.1 (Proteome Software Inc., Portland, OR) (28.Searle B.C. Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies.Proteomics. 2010; 10: 1265-1269Crossref PubMed Scopus (396) Google Scholar). Peptides and proteins were identified using the Peptide Prophet algorithm (29.Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3897) Google Scholar), using a probability cut-off of 95% (peptides) or 99% probability (proteins), and contained at least two identified peptides (proteins) (30.Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3631) Google Scholar). Proteins containing similar peptides that could not be differentiated based on MS/MS analysis were grouped to satisfy the principles of parsimony. A false discovery rate of <0.1% was calculated using protein identifications validated using the Scaffold program (v.4.2.1). Proteins were classified according to GO categories using the program Blast2Go (31.Conesa A. Gotz S. Garcia-Gomez J.M. Terol J. Talon M. Robles M. Blast2GO: a universal tool for annotation, visualization, and analysis in functional genomics research.Bioinformatics. 2005; 21: 3674-3676Crossref PubMed Scopus (8593) Google Scholar) and Pfam using HMMER v3.1b1 (32.Finn R.D. Clements J. Eddy S.R. 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