Rapid and comprehensive discovery of unreported shellfish allergens using large-scale transcriptomic and proteomic resources

Abstract

Discovery of allergens in various food and inhalant sources is central to our understanding of the molecular mechanisms of allergic reactions. Allergen characterization is the most important underlying factor for better patient management and the design and development of novel immunotherapeutics.1Yu W. Freeland D.M.H. Nadeau K.C. Food allergy: immune mechanisms, diagnosis and immunotherapy.Nat Rev Immunol. 2016; 16: 751-765Crossref PubMed Scopus (284) Google Scholar Of the “Big eight” allergen food groups, shellfish presents a unique challenge in terms of allergen discovery due to the large number and diversity of consumed species, leading to heterogeneity of allergen structure and cross-reactivity among various sources. At present, 31 crustacean allergens have been officially registered in the World Health Organization and International Union of Immunological Societies Allergen Nomenclature Database as compared with 4 mollusk allergens due to pitfalls in current allergy discovery approaches. Cosensitization of patients with crustacean and mollusk allergy is often described; however, the current diagnostic approaches to manage these patients are not based on sufficient molecular knowledge of these shellfish allergens. In this study, an innovative strategy was used for the identification of novel cross-reactive allergenic proteins in the Pacific Oyster, using a combined approach of high-throughput screening of the genome and proteome along with comparative confirmation using protein family domains and allergen databases, identifying allergens with high probability. The study design (see Fig E1 in this article's Online Repository) and methods are described in this article's Online Repository at www.jacionline.org. Initially, we used a bioinformatics approach for identifying potential allergens in the Pacific Oyster proteome. To select promising candidates, we first enumerated proteins from the Pacific Oyster genome–derived proteome (25,982 proteins). Subsequently, allergen protein domains were detected by interrogating Hidden Markov Model profiles using HMMER3.2Eddy S.R. Accelerated profile HMM searches.PLoS Comput Biol. 2011; 7: e1002195Crossref PubMed Scopus (1725) Google Scholar The term “allergen protein domain” is used to define protein families that include allergenic proteins. These domains were derived from the Pfam database version 29.0,3Finn R.D. Coggill P. Eberhardt R.Y. Eddy S.R. Mistry J. Mitchell A.L. et al.The Pfam protein families database: towards a more sustainable future.Nucleic Acids Res. 2016; 44: D279-D285Crossref PubMed Google Scholar which accounted for only 273 of the known 16,295 protein domains (2%). We discovered that 2504 Pacific Oyster proteins are associated with at least 1 of the known allergen protein domains. We next used the full-length protein sequence alignment to these 2504 proteins using the BLASTP program against 2117 known allergens to identify proteins that are homologous to the known allergens. A minimum of 50% shared identity and an upper threshold of E = 10−7 were used as display limits of alignment.4Goodman R.E. Ebisawa M. Ferreira F. Sampson H.A. van Ree R. Vieths S. et al.AllergenOnline: a peer-reviewed, curated allergen database to assess novel food proteins for potential cross-reactivity.Mol Nutr Food Res. 2016; 60: 1183-1198Crossref PubMed Scopus (0) Google Scholar Using these 2 criteria, we identified 95 proteins that have significant sequence identity with known allergenic proteins. Of these, 22 proteins were categorized as “very likely allergenic” with an amino acid identity of 70% or more with known allergens and 73 proteins as “likely allergenic” with amino acid identities between 50% and 70% (Fig 1, A.I; see Table E1 in this article's Online Repository at www.jacionline.org). Furthermore, a comprehensive analysis of the expression of allergen coding genes, across different developmental stages of the oyster and different tissues, was conducted on the basis of currently available transcriptomic data from the oyster genome project.5Zhang G. Fang X. Guo X. Li L. Luo R. Xu F. et al.The oyster genome reveals stress adaptation and complexity of shell formation.Nature. 2012; 490: 49-54Crossref PubMed Scopus (876) Google Scholar As illustrated in Fig 1, A.II, and Fig E2 in this article's Online Repository at www.jacionline.org, potential allergens were differentially expressed across developmental stages and various tissues of the Pacific Oyster. For example, both isoforms of tropomyosin are highly expressed in the adult stage, specifically in the adductor muscle and mantle tissues. In contrast, heat shock protein HSP 68 is expressed at a higher level in early developmental stages as compared with the adult oyster. The next step involving allergenomics included the identification of IgE-binding proteins using serum of 5 mollusk-sensitized patients. These patients reported clinical reactivity to various shellfish species including oyster, mussel, prawn, and lobster as well as reactivity to fish and house dust mite (HDM; see Table E2 in this article's Online Repository at www.jacionline.org). Protein extracts from raw and heated Pacific Oyster were separated by 2-dimensional electrophoresis, and visualized using Coomassie Blue or IgE antibody binding analyzed by immunoblotting (Fig 1, B). Twenty-two IgE-reactive spots were excised from the resolved raw oyster extract and 5 spots from the heated extract (see Fig E3 in this article's Online Repository at www.jacionline.org), followed by in-gel tryptic digestion and mass spectrometric analysis. We identified a total of 332 and 26 proteins in the IgE-reactive spots of the raw and heated extracts, respectively. Some proteins were discovered in several different spots, due to polymorphisms (source data are available via ProteomeXchange with identifier PXD006624). The composition of water-soluble proteins in the raw and heated oyster extracts was identified using shotgun mass spectrometry. We identified 1097 proteins in the raw extract and 133 proteins in the heated extract. The comparative analysis of relative protein abundance, using the emPAI scores, indicated that sarcoplasmic calcium-binding protein is the most abundant water-soluble protein in both extracts, followed by arginine kinase and fatty acid binding protein in the raw extract. Other known heat-stable shellfish allergens identified in the heated extracts are tropomyosin and myosin light chain (Fig 1, C). Most of the allergens that have been characterized in commonly consumed shellfish species have been demonstrated to be heat stable.6Kamath S.D. Rahman A.M. Voskamp A. Komoda T. Rolland J.M. O'Hehir R.E. et al.Effect of heat processing on antibody reactivity to allergen variants and fragments of black tiger prawn: a comprehensive allergenomic approach.Mol Nutr Food Res. 2014; 58: 1144-1155Crossref PubMed Scopus (88) Google Scholar The comparative evaluation of in silico bioinformatics analysis with the allergenomic and proteomic data generated confirms that 24 proteins in the raw extract and 4 in the heated extract were identified using all 3 methods (Fig 1, D, and Table I). These allergenic proteins have a high probability to elicit immunological cross-reactivity in oyster-allergic patients to other organisms containing the same or highly similar allergens. For example, triosephosphate isomerase and 78-kDa glucose-regulated protein share 76% and 65% amino acid identity with homologous dust mite allergens, respectively, potentially responsible for clinical mollusk-mite cross-reactivity, previously reported during mite immunotherapy.7Tsapis M. Chabane H. Teychene A.M. Laurent M. Roland P.N. de Pontual L. Fatal anaphylaxis after snail ingestion in a child after 3 years of house dust mite immunotherapy.Rev Fr Allergol. 2013; 53: 436-438Crossref Scopus (2) Google Scholar Furthermore, oyster enolases share 61% to 71% amino acid identity with enolase from fish, latex, grass pollen, and fungi. However, clinical reactivity due to enolase of oyster or these other allergen sources has not yet been reported. The importance of early identification of sensitizing allergens has been corroborated by several clinical studies, including the recently described chicken-fish syndrome.8Kuehn A. Codreanu-Morel F. Lehners-Weber C. Doyen V. Gomez-Andre S.A. Bienvenu F. et al.Cross-reactivity to fish and chicken meat—a new clinical syndrome.Allergy. 2016; 71: 1772-1781Crossref PubMed Scopus (52) Google Scholar Similarly, Chan et al9Chan T.F. Ji K.M. Yim A.K. Liu X.Y. Zhou J.W. Li R.Q. et al.The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens.J Allergy Clin Immunol. 2015; 135: 539-548Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar conducted the first genomic analysis of HDMs (Dermatophagoides farinae) and developed a combined genomic-transcriptomic-proteomic approach to elucidate previously unreported HDM allergens, identifying 12 new allergens also present in nonmite species.Table IProteins identified across all 3 methods with their matched allergens source and routes of sensitizationNo.EntryProtein nameHomologous allergen in the IUIS database∗Only homologous allergen with the highest identity has been displayed.Amino acid (aa) identity (%)Overlap (aa)E valueOrganismRoute of sensitizationSource1B7XC66Tropomyosin†Proteins identified in both raw and heated extract.Hel as 175.72844.00 × 10−133Helix aspersa (Brown garden snail)IngestionAnimal2K1PCV6Triosephosphate isomeraseCra c 874.03772.00 × 10−41Crangon crangon (North sea shrimp)IngestionAnimal3K1PJ59Triosephosphate isomeraseDer f 2573.371696.00 × 10−92Dermatophagoides farinae (HDM)InhalationAnimal4K1QX37EnolaseThu a 272.833570Thunnus albacares (Yellowfin tuna)IngestionAnimal5K1Q350Glyceraldehyde-3-phosphate dehydrogenaseTri a 3471.213307.00 × 10−173Triticum aestivum (Wheat)IngestionPlant6Q75W4978-kDa glucose- regulated proteinCor a 1071.046180Corylus avellana (European hazelnut)InhalationPlant7K1QTC1Paramyosin‡Paramyosin currently not registered in the IUIS database has more than 50% identity. The alignment was determined against allergens from the AllergenOnline database.—68.597990Haliotis discus discus (Abalone)IngestionAnimal8K1R8R6Fructose-bisphosphate aldolaseSal s 367.313644.00 × 10−177Salmo salar (Atlantic salmon)IngestionAnimal9K1RTQ6Fructose-bisphosphate aldolaseSal s 366.673631.00 × 10−178Salmo salar (Atlantic salmon)IngestionAnimal10K1PNQ5Heat shock protein HSP 90-alpha 1Asp f 1266.264120Aspergillus fumigatusInhalationFungi11K1QNV6Tropomyosin†Proteins identified in both raw and heated extract.Tod p 165.312713.00 × 10−84Todarodes pacificus (Squid)IngestionAnimal12K1R266Retinal dehydrogenase 1†Proteins identified in both raw and heated extract.Tyr p 35614721.00 × 10−170Tyrophagus putrescentiae (Storage mite)InhalationAnimal13K1QNT7Aldehyde dehydrogenase, mitochondrialTyr p 35604821.00 × 10−180Tyrophagus putrescentiae (Storage mite)InhalationAnimal14K1QVK0TransaldolaseFus p 459.23261.00 × 10−125Fusarium proliferatumInhalationFungi15K1QVG5Retinal dehydrogenase 1†Proteins identified in both raw and heated extract.Tyr p 35594741.00 × 10−169Tyrophagus putrescentiae (Storage mite)InhalationFungi16K1PLF9Arginine kinaseBomb m 159.133453.00 × 10−147Bombyx mori (Silkworm moth)IngestionAnimal17K1Q3F4Inorganic pyrophosphataseDer f 3258.242611.00 × 10−113Dermatophagoides farinae (HDM)InhalationAnimal18K1Q9Z4Aldehyde dehydrogenaseTyr p 35561958.00 × 10−65Tyrophagus putrescentiae (Storage mite)InhalationAnimal19K1P9D0Stress-70 protein, mitochondrialPen c 1955.224313.00 × 10−163Penicillium citrinumInhalationFungi20K1QX26EndoplasminAsp f 1254.041989.00 × 10−61Aspergillus fumigatusInhalationFungi21K1Q7T5Protein disulfide-isomeraseAlt a 452.27443.00 × 10−11Alternaria alternataInhalationFungi22K1Q5P7Peptidyl-prolyl cis-trans isomeraseCat r 152.171615.00 × 10−53Catharanthus roseus (Madagascar periwinkle)InhalationPlant23K1R4Z3Malate dehydrogenase, mitochondrialMala f 451.432801.00 × 10−91Malassezia furfurInhalationFungi24K1Q6X5Protein disulfide-isomeraseAlt a 450681.00 × 10−15Alternaria alternataInhalationFungiThe proteins are sorted on the basis of amino acid sequence identity with their homologous allergens in descending order.IUIS, International Union of Immunological Societies.∗ Only homologous allergen with the highest identity has been displayed.† Proteins identified in both raw and heated extract.‡ Paramyosin currently not registered in the IUIS database has more than 50% identity. The alignment was determined against allergens from the AllergenOnline database. Open table in a new tab The proteins are sorted on the basis of amino acid sequence identity with their homologous allergens in descending order. IUIS, International Union of Immunological Societies. In summary, our innovative methodological approach using biochemical and computational tools in addition to antibody reactivity was successful in identifying 24 previously unreported allergens from more than 25,000 proteins of the Pacific Oyster. This is the first study to demonstrate the presence of multiple novel allergens in any shellfish species. Some of these are common to very different allergen sources incorporating animal, including fish and mites, as well as plant allergens from pollen, latex, and fungi. Importantly, we demonstrated that these allergenic proteins identified are reactive to shellfish-allergic patients’ IgE antibodies under in vitro conditions. The rapid and comprehensive analysis of unreported allergenic proteins fills a major gap in the current management of patients at high risk of concurrent reactivity to diverse allergen sources. To identify potential allergens of Pacific Oyster, full-length sequence alignment of oyster proteins was carried out against a repertoire of known allergens. For this purpose, 2 data sets were assembled. The first data set contained a FASTA file of 25,982 genome-derived proteins of the Pacific OysterE1Zhang G. Fang X. Guo X. Li L. Luo R. Xu F. et al.The oyster genome reveals stress adaptation and complexity of shell formation.Nature. 2012; 490: 49-54Crossref PubMed Scopus (1542) Google Scholar collected from the UniProt database (Proteome ID UP000005408, last modification October 9, 2016). The second data set contained 2117 allergen sequences compiled from 2 main allergen databases: the World Health Organization and International Union of Immunological Societies Allergen Nomenclature (http://www.allergen.org/)E2Radauer C. Nandy A. Ferreira F. Goodman R.E. Larsen J.N. Lidholm J. et al.Update of the WHO/IUIS Allergen Nomenclature Database based on analysis of allergen sequences.Allergy. 2014; 69: 413-419Crossref PubMed Scopus (128) Google Scholar and the Food Allergy Research and Resource Program (Version 16, http://www.allergenonline.org/).E3Goodman R.E. Ebisawa M. Ferreira F. Sampson H.A. van Ree R. Vieths S. et al.AllergenOnline: a peer-reviewed, curated allergen database to assess novel food proteins for potential cross-reactivity.Mol Nutr Food Res. 2016; 60: 1183-1198Crossref PubMed Scopus (96) Google Scholar Genbank accession IDs of all allergenic proteins were collected from these databases and the IDs uploaded in the Batch Entrez menu on the National Center for Biotechnology Information Web site to obtain the sequence of the protein and remove duplicate proteins. Before the sequence alignment, we filtered the oyster proteome by the Pfam domains containing allergens. The latest distribution of protein families from the allergen data set was defined by running the hmmscan programE4Eddy S.R. Accelerated profile HMM searches.PLoS Comput Biol. 2011; 7: e1002195Crossref PubMed Scopus (3162) Google Scholar against the Pfam database (version 29.0).E5Finn R.D. Coggill P. Eberhardt R.Y. Eddy S.R. Mistry J. Mitchell A.L. et al.The Pfam protein families database: towards a more sustainable future.Nucleic Acids Res. 2016; 44: D279-D285Crossref PubMed Scopus (3585) Google Scholar The BLASTP program was used to align the Pacific Oyster proteins and the repertoire of known allergens using a cutoff E value of 10−7 and sequence identity of more than 50%. The expression levels of potential allergen genes were analyzed from the available RNA sequencing data from the oyster genome project.E1Zhang G. Fang X. Guo X. Li L. Luo R. Xu F. et al.The oyster genome reveals stress adaptation and complexity of shell formation.Nature. 2012; 490: 49-54Crossref PubMed Scopus (1542) Google Scholar The expression profiles were analyzed from 2 developmental stages (spat and juvenile) and from 10 adult organs: the adductor muscle, the digestive gland, the female gonad, the male gonad, the gill, the hemocyte, the labial palp, the outer mantle, the inner mantle, and the remaining tissue. The expression levels were calculated by RPKM (Reads Per Kilobase of transcript per Million mapped reads). Fresh oyster specimens were purchased from the local market and stored at −20°C before use. Protein extracts were prepared according to the method of Kamath et alE6Kamath S.D. Abdel Rahman A.M. Komoda T. Lopata A.L. Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies.Food Chem. 2013; 141: 4031-4039Crossref PubMed Scopus (130) Google Scholar with slight modification. The meat of the oyster was finely cut and homogenized in PBS (pH 7.2) for 10 minutes, using an Ultra turrax homogenizer (IKA, Staufen, Germany). After gentle shaking at 4°C for 3 hours and centrifugation at 20,000g for 20 minutes, supernatants were clarified through a glass fiber filter, followed by filtration through a 0.45-μm membrane filter (Sartorius AG, Goettingen, Germany) and stored at −80°C before further use. To produce heated protein extracts, the meat was heated in PBS at 95°C to 100°C for 20 minutes instead of heating the raw extract, to mimic the way consumers are exposed to food allergens. The meat was removed after cooling, and the proteins were extracted using the same method as described above. The total protein content of the extracts was quantified using the bicinchoninic acid assay (Pierce, Rockford, Ill) following the manufacturer's instructions. A prediluted set of BSA was used as protein standards (Pierce). Total protein of the Pacific Oyster raw and heated extracts was identified after trypsin in-gel digestion. Briefly, 20 μg of the extracts was loaded onto a 12% polyacrylamide gel and run at 170 V for 1 hour. The gels were cut into pieces and washed with 25 mM ammonium bicarbonate. After being dried using a vacuum dryer, the gels were reduced by 20 mM dithiothreitol (DTT) at 65°C for 1 hour and alkylated by 50 mM iodoacetamide for 40 minutes at 37°C in the dark. Gel pieces were washed and dried using a SpeedVac. Dried gel pieces were rehydrated with 20 ng/μL of trypsin dissolved in 40 mM ammonium bicarbonate and 10% acetonitrile for 1 hour at room temperature and subsequently incubated overnight at 37°C. The digested proteins were acidified using 0.1% formic acid and the peptides were concentrated on a SpeedVac and subjected to Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis. Five subjects with a convincing clinical history of allergic reactivity to shellfish and 1 nonatopic subject were recruited from the Alfred Hospital Allergy Clinic, Melbourne, Victoria, Australia. Skin prick testing and oral challenge with mollusk extracts were not conducted routinely in these patients, in keeping with the clinicians' preference for safer serum specific allergen IgE testing in adult patients due to comorbidities, together with clinical history of reactions on exposure. Ethics approval for this study was granted by James Cook University's Ethics Committee (project no. H4313) in collaboration with the Alfred Hospital (project no. 192/07) and Monash University's Ethics Committees (MUHREC CF08/0225). The proteins in the oyster extracts were separated by 2-dimensional gel electrophoresis. Proteins were first lyophilized and resuspended in 8 mol urea, 2% CHAPS, 50 mM DTT, and 0.2% (w/v) Biolyte 3/10 ampholytes buffer. The extract was subjected to isoelectric focusing using 3 to 10 NL pH range 12% ReadyStrip IPG Strips (Bio-Rad, Hercules, Calif), as per the manufacturer's instructions. Briefly, 185 μL of rehydration buffer containing 200 μg of raw extract or 100 μg of heated extract was loaded onto the IPG tray, and a strip was gently placed side down onto the sample and left to incubate overnight at room temperature. Isoelectric focusing was conducted using a PROTEAN IEF cell (Bio-Rad) with a maximum current of 50 μA/strip. After focusing, the IPG strips were equilibrated with equilibration buffer (ES) 1 and ES 2. The ES 1 contained 6 mol urea, 2% SDS, 20% glycerol, 0.375 mol Tris-HCl, pH 8.8, and 2% (w/v) DTT, and the ES 2 contained the same solution as ES 1 except that it contained 4% iodoacetamide instead of DTT. The strip was then washed with SDS-PAGE running buffer and laid on top of a 12% polyacrylamide gel. The gels were run at 170 V until bromophenol blue dye reached the bottom of each gel. Gels were either stained with Coomassie Brillian Blue R-250 or the separated proteins were transferred to polyvinylidene difluoride membrane. Protein transfer was performed using the Semi-dry TransBlot Apparatus (Bio-Rad). After blocking with 5% (w/v) skim milk powder in PBS with 0.05% Tween, the membrane was incubated with a serum pool from 5 shellfish-allergic patients at a 1:20 dilution overnight at 4°C with shaking. The membrane was subsequently incubated with 1:10,000 dilution of rabbit antihuman IgE (Dako, Glostrup, Denmark) followed by 1:10,000 dilution of horseradish peroxidase–conjugated goat anti-rabbit antibodies (Promega, Madison, Wis). Specific IgE binding was detected by chemiluminescence and exposed to photographic film (GE Healthcare Biosciences, Buckinghamshire, UK) to visualize the antibody-binding protein spots. Serum from a nonatopic donor was used as a negative control. IgE-reactive spots were annotated using the proteome map and corresponding bands were cut, tryptic digested, and analyzed using mass spectrometry. The LC-MS/MS was carried out on an LTQ Orbitrap Elite (Thermo Scientific) with a nanoESI interface in conjunction with an Ultimate 3000 RSLC nanoHPLC (Dionex Ultimate 3000) at the Bio21 Institute, Melbourne, Australia. The LC system was equipped with an Acclaim Pepmap nano-trap column (Dionex-C18, 100 Å, 75 μm × 2 cm) and an Acclaim Pepmap RSLC analytical column (Dionex-C18, 100 Å, 75 μm × 50 cm). The tryptic peptides were injected into the enrichment column at an isocratic flow rate of 5 μL/min of 3% v/v CH3CN containing 0.1% v/v formic acid for 5 minutes before the enrichment column was switched in-line with the analytical column. The eluents were 0.1% v/v formic acid (solvent A) and 100% v/v CH3CN in 0.1% v/v formic acid (solvent B). The flow gradient was (1) 0 to 5 minutes at 3% B, (2) 5 to 25 minutes at 3% to 25% B, (3) 25 to 27 minutes at 25% to 40% B, (4) 27 to 29 minutes at 40% to 80% B, (5) 29 to 31 minutes at 80% B, (7) 31 to 32 minutes at 80%-3% B, and (8) 32 to 38 minutes at 3% B. The LTQ Orbitrap Elite spectrometer was operated in the data-dependent mode with nanoESI spray voltage of 1.8 kV, a capillary temperature of 250°C, and S-lens RF value of 55%. All spectra were acquired in the positive mode with full-scan MS spectra from m/z 300 to 1650 in the FT mode at 240,000 resolution. Automated gain control was set to a target value of 1.0−6 and a lock mass of 445.120025 was used. The top 20 most intense precursors were subjected to rapid collision–induced dissociation with a normalized collision energy of 30 and activation q of 0.25. A dynamic exclusion of 30 seconds was applied for repeated precursors. All MS/MS files were analyzed using Mascot v2.4 against the in-house database of the oyster proteome downloaded from the UniProt, supplemented with the common Repository of Adventitious Proteins sequences. Search parameters were as follows: precursor mass tolerance of 200 ppm and fragment mass tolerance of 0.6 Da (CID). Carbamidomethyl (C) was set as a fixed modification, and oxidation (M) and deamidated (NQ) were set as variable modifications. Trypsin with a maximum of 3 missed cleavages was used as the cleavage enzyme. Scaffold (version Scaffold_4.7.3, Proteome Software, Inc, Portland, Ore) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithmE7Keller 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 with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.E8Nesvizhskii 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 (3633) Google Scholar Proteins that contained similar peptides and could not be differentiated on the basis of MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDEE9Vizcaíno J.A. Csordas A. del-Toro N. Dianes J.A. Griss J. Lavidas I. et al.2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: D447-D456Crossref PubMed Scopus (2782) Google Scholar partner repository with the data set identifier PXD006624 and 10.6019/PXD006624.Fig E2Expression profiles of genes encoding “likely allergens” across different development stages and different tissues of the Pacific Oyster. The transcriptome expression data were obtained from GigaDB database under data set number 100030. Gene expression levels were measured by RPKM (Reads Per Kilobase per Million mapped reads).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Coomassie-stained 2D-PAGE of the proteins from the raw and heated extracts of the Pacific Oyster. Spot numbers indicated on the gel correspond to IgE-reactive spots that were subjected to LC/MS mass spectrometry analysis. 2D, Two-dimensional.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Identified potential allergens of Pacific Oyster from the in silico analysisProteinGene nameBest matched allergenAmino acid identity (%)Very likely allergenic (amino acid identity ≥70%) TropomyosinCGI_10013163Tropomyosin (Crassostrea gigas)92.94 TropomyosinCGI_10013164Tropomyosin, partial (Crassostrea virginica)86.67 Tubulin alpha chainCGI_10002456Der f 33 allergen (Mite)85.71 Tubulin alpha-1C chainCGI_10002455Der f 33 allergen (Mite)83.2 Tubulin alpha-3 chainCGI_10018930Der f 33 allergen (Mite)82.43 Tubulin alpha-1C chainCGI_10024998Der f 33 allergen (Mite)81.8 Tubulin alpha-1C chainCGI_10007570Der f 33 allergen (Mite)81.53 Tubulin alpha-1C chainCGI_10024999Der f 33 allergen (Mite)81.35 78-kDa glucose-regulated proteinCGI_10015492Aed a 8 (Mosquito)81 Tubulin alpha-1C chainCGI_10002454Der f 33 allergen (Mite)80 Tubulin alpha-1C chainCGI_10008247Der f 33 allergen (Mite)77.7 Tubulin alpha-1A chainCGI_10007571Der f 33 allergen (Mite)77.63 Fructose-bisphosphate aldolaseCGI_10019801Thu a 3 (Tuna)74.29 Heat shock protein 70 B2CGI_10010646Der f 28 allergen (Mite)74.27 Heat shock protein 70 B2CGI_10010647Der f 28 allergen (Mite)74.27 Triosephosphate isomeraseCGI_10003538Triosephosphate isomerase (Shrimp)74.03 Heat shock protein 68CGI_10002594Der f 28 allergen (Mite)73.96 Triosephosphate isomeraseCGI_10003539Der f 25 allergen (Mite)73.37 Heat shock protein 70 B2CGI_10003417Der f 28 allergen (Mite)73.13 EnolaseCGI_10022154Enolase (Tunas)72.83 Fructose-bisphosphate aldolaseCGI_10025556Thu a 3 (Tuna)72.22 Glyceraldehyde-3-phosphate dehydrogenaseCGI_10010974Glyceraldehyde-3-phosphate dehydrogenase (Wheat)71.21 Peptidyl-prolyl cis-trans isomerase E (PPIase E)CGI_10026365Der f 6 (Mite)70.19Likely allergenic (amino acid identity ≥50%, but <70%) FerritinCGI_10016317Ferritin (Mite)69.92 ParamyosinCGI_10001653Paramyosin (Abalone)68.59 Arginine kinaseCGI_10021480Arginine kinase (Octopus)66.96 Arginine kinaseCGI_10021483Arginine kinase (Octopus)66.67 Heat shock protein HSP 90-alpha 1CGI_10017621Asp f 12 (Fungus)66.26 FerritinCGI_10021660Ferritin (Mite)66.06 Cytochrome cCGI_10012574Cur l 3 (Fungus)66.02 Superoxide dismutase (Cu-Zn)CGI_10017958Ole e 3 (Olive tree)66 Peptidyl-prolyl cis-trans isomerase 6CGI_10022249Cyclophilin (Carrot)65.96 FerritinCGI_10027591Ferritin (Mite)65.58 78-kDa glucose-regulated proteinCGI_10008834Der f 28 (Mite)65.3 78-kDa glucose-regulated proteinCGI_10027395Der f 28 (Mite)65.25 Arginine kinaseCGI_10024056Arginine kinase (Octopus)64.65 Inorganic pyrophosphataseCGI_10027722Der f 32 (Mite)64.24 Fructose-bisphosphate aldolaseCGI_10000078Sal s 3 (Salmon)64.12 60S ribosomal protein L3CGI_10010529Asp f 23 (Fungus)63.96 Arginine kinaseCGI_10021482Arginine kinase (Octopus)63.83 Heat shock protein 68CGI_10002823Der f 28 (Mite)63.34 40-kDa peptidyl-prolyl cis-trans isomeraseCGI_10015504Cyclophilin (Carrot)62.72 Peptidyl-prolyl cis-trans isomeraseCGI_10013880Cyclophilin (Carrot)61.76 Plasma kallikreinCGI_10016607Der f 3 (Mite)61.7 60S ribosomal protein L3CGI_10012282Asp f 23 (Fungus)60.76 Arginine kinaseCGI_10021481Arginine kinase (Octopus)60.6 Tubulin alpha chainCGI_10018903Der f 33 (Mite)60.49 78-kDa glucose-regulated proteinCGI_10018425Der f 28 (Mite)59.9 Eukaryotic translation initiation factor 3 subunit I (fragment)CGI_10025943For t 1 (Midges)59.88 Calcium-binding atopy-related autoantigen 1CGI_10026057Hom s 4 (Human)59.3 TransaldolaseCGI_10002311Fus p 4 (Fungus)59.2 Peptidyl-prolyl cis-trans isomerase BCGI_10023851Cyclophilin (Carrot)58.7 Peptidyl-prolyl cis-trans isomeraseCGI_10023850Asp f 27 (Fungus)58.33 Aldehyde dehydrogenase, mitochondrialCGI_10012671Cla h 10 (Fungus)58.02 ThioredoxinCGI_10021611Mala s 13 (Yeast)58 Peptidyl-prolyl cis-trans isomeraseCGI_10024975Cyclophilin (Carrot)57.63 Thioredoxin domain-containing protein 5CGI_10009327Alt a 4 (Fungus)57.45 60S acidic ribosomal protein P1CGI_10009326Alt a 12 (Fungus)57.14 NK-tumor recognition proteinCGI_10007438Asp f 27 (Fungus)56.8 Thaumatin-like protein 1aCGI_10012508Pathogenesis related protein 5 (Apple)55.62 Superoxide dismutaseCGI_10017307Pis v 4 (Pistachio)55.61 Peptidyl-prolyl cis-trans isomeraseCGI_10006179Asp f 11 (Fungus)55.56 Peptidylprolyl isomerase domain and WD repeat-containing protein 1CGI_10011521Mala s 6 (Yeast)55.47 Stress-70 protein, mitochondrialCGI_10016162Pen c 19 (Fungus)55.22 Peptidyl-prolyl cis-trans isomerase-like 6CGI_10024382Cat r 1 (Periwinkle)55.22 U4/U6.U5 tri-snRNP-associated protein 1CGI_10021218Hom s 1 (Human)54.96 Alpha-amylaseCGI_10022190Bla g 11 (Cockroach)54.37 Alpha-amylaseCGI_10022189Bla g 11 (Cockroach)54.18 EndoplasminCGI_10025730Asp f 12 (Fungus)54.04 CalmodulinCGI_10006247Amb a 9 (Ragweed)53.7 CalmodulinCGI_10014525B1 protein allergen (Bermuda grass)53.57 Aldehyde dehydrogenaseCGI_10021688Cla h 10 (Fungus)52.82 Retinal dehydrogenase 1CGI_10026868Cla h 10 (Fungus)52.33 Protein disulfide-isomeraseCGI_10011652Alt a 4 (Fungus)52.27 Collagen alpha-3(VI) chainCGI_10015798collagen alpha (Bovine)52.27 Peptidyl-prolyl cis-trans isomerase BCGI_10027458Cat r 1 (Periwinkle)52.17 78-kDa glucose-regulated proteinCGI_10011272Der f 28 (Mite)52.1 ThioredoxinCGI_10003765Asp f 28 (Fungus)51.85 Uncharacterized proteinCGI_10016401Der f 3 (Mite)51.85 Transcription elongation factor 1-like proteinCGI_10026699Tri a 45 (Wheat)51.81 Alpha-amylaseCGI_10023778Bla g 11 (Cockroach)51.75 Malate dehydrogenase, mitochondrialCGI_10015004Mala f 4 (Yeast)51.43 CalmodulinCGI_10022491Tyr p 24 (Mite)51.32 Retinal dehydrogenase 1CGI_10026867Cla h 10 (fungus)51.27 Peptidyl-prolyl cis-trans isomeraseCGI_10005876Mala s 6 (Yeast)51.16 CalmodulinCGI_10011301Par j 4 (Weed)50.94 CalmodulinCGI_10006481Syr v 3 (Common lilac)50.85 Alpha-amylaseCGI_10023781Bla g 11 (Cockroach)50.51 Elongation factor 1-betaCGI_10021397Pen c 22 (Fungus)50.44 CalmodulinCGI_10002924B1 protein allergen (Bermuda grass)50 Calmodulin-like protein 12CGI_10004114Putative Cup a 4 allergen (Cypress)50 CalmodulinCGI_10011293Polcalcin (Artemisia vulgaris)50 SupervillinCGI_10014153Villin 2 (Tobacco)50 CalmodulinCGI_10017056Bla g 6 (Cockroach)50 Protein disulfide-isomeraseCGI_10026048Alt a 4 (Fungus)50 Open table in a new tab Table E2Demographic characteristics of patients recruited for this studySubjectSexAge (y)Total IgE (kU/L)Specific IgE (ImmunoCAP) (kU/L)Skin prick testOther allergiesOysterShrimpHDMOysterShrimpHDM1M509762.049.0313.60NTNT12 mmTuna, cod2F284610.110.3654.8NTNT6 mmNT3M43194NT1.410.353 mm10 mm10 mmNT4F38283.759.822.66NTNT0 mmNT5M381831.046.8431.70NTNTNTNTF, Female; M, male; NT, not tested. Open table in a new tab F, Female; M, male; NT, not tested.