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  1. 1. Article pubs.acs.org/jprComprehensive Proteomics Approach in Characterizing andQuantifying Allergenic Proteins from Northern Shrimp: TowardBetter Occupational Asthma PreventionAnas M. Abdel Rahman,*,†,‡ Sandip D. Kamath,§ Sébastien Gagné,∥ Andreas L. Lopata,§and Robert Helleur‡†Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada‡Department of Chemistry, Memorial University of Newfoundland, St. John’s, NL, Canada§School of Pharmacy and Molecular Science and Center of Biodiscovery and Development of Molecular Therapeutics, James CookUniversity, Australia∥ Institut de Recherche Robert-Sauvé en Santé et Sécurité au Travail, Montreal, Quebec, Canada S * Supporting Information ABSTRACT: Occupational asthma is a major chronic health dilemma among workers involved in the seafood industry. Several proteins notoriously known to cause asthma have been reported in dixerent seafood. This work involves the application of an allergeno- mics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and proxled by gel electrophoresis. Allergenic proteins were identixed based on their reactivity to patient sera and were structurally identixed using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most signixcant allergens. Multiple proteolytic enzymes enabled 100% coverage of the sequence of shrimp tropomyosin by tandem mass specrometry. Only partial sequence coverage was obtained, however, for the shrimp allergen arginine kinase. Signature peptides, for both tropomyosin and arginine kinase, were assigned and synthesized for use in developing the multiple reaction monitoring tandem mass spectrometric method. Subsequently, air samples were collected from a shrimp processing plant and two aerosolized proteins quantixed using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m 3 for tropomyosine and arginine kinase, respectively. Tropomyosine is much more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. The present study shows that mass spectrometric analysis is a sensitive and accurate tool in identifying and quantifying aerosolized allergens. KEYWORDS: allergenomics, occupational asthma, seafood allergen, mass spectrometry, proteomics, northern shrimp, aeroallergen, environmental proteomics■ INTRODUCTIONFood allergy is one of the most common causes of anaphylaxis, Double-blind placebo controlled food challenges (DBPCFCs) were performed to determine threshold shrimp doses forwhich is responsible for hundreds of fatalities annually worldwide. 1 individuals with shrimp allergies. The threshold doses rangedShrimp allergy axects about 2% of the general population world- from 14 to 16 g of shrimp equivalent to 32 mg of protein andwide.2−4 In recent decades, seafood consumption has dramatically from 5 to 600 mg of protein for dixerent xsh. 4−7 Seafood allergenincreased as a healthier diet choice, which in turn has increased the quantixcation was initiated in 1997 by Lehrer’s group using anumber of people engaged in the seafood industry. These workers sandwich Enzyme-linked immunosorbent assay (ELISA) approachare frequently exposed to aeroallergens that cause type-I hyper- for targeting brown shrimp tropomyosin (TM). 8 The detectionsensitivity. Northern shrimp (Pandalus borealis), caught in the limit was 4 ng/mL, and the assay was applied to detect TM inNorth Atlantic and North Pacixc oceans, are the most commonly dixerent crustacean species such as crab and lobster. Recently, theconsumed shrimp in North America. The lack of molecular data method was optimized to evaluate the level of crustacean majoron allergenic proteins from most seafood species requires thedevelopment of comprehensive strategies to study the biochemicalcharacteristics of these allergens. Received: August 8, 2012 Published: December 26, 2012 © 2012 American Chemical Society 647 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  2. 2. Journal of Proteome Research ArticleFigure 1. Allergenomics strategy for allergen discovery using functional proteomics followed by a mass spectrometric approach for molecularcharacterization.17allergen, TM, in processed food9,10 with a limit of detection of ethylenediaminetetraacetic acid (EDTA), formic acid (FA),about 2.5 mg/kg. 11 ammonium bicarbonate, o-ethylisourea hemisulfate, ammonium- Although TM is the major crustacean allergen responsible for hydroxide, horseradish peroxidase (HRP), chemiluminescentingestion-related allergic reactions,12 other allergens were substrate, sodium dodecylsulfate (SDS), ammonium formate, andidentixed and characterized such as arginine kinase (AK), 13,14 α-cyano-4-hydroxycinamic acid (HCCA) matrix were purchasedsarcoplasmic calcium-binding protein (SCBP), 15,16 and myosin- from Sigma-Aldrich (St. Louis, MO, USA). The Bradford assay kitlight chain (MLC).17,18 and PVDF immunoblot membranes were purchased from BioRad This study introduces a functional proteomics strategy (Hercules, CA, USA), and dialysis bags were purchased from(allergenomic) to evaluate the potential allergenic proteins in north- Fischer Scientixc (Roncho Dominguez, CA, USA). ZipTip C18ern shrimp (NS) as summarized in Figure 1.19 Sera from sensitized xlters were purchased for desalting from Millipore Corporationpatients were used to evaluate the proteomics proxle of NS which (Bedford, MA, USA). Powdered skimmed milk was purchasedwas subsequently identixed using mass spectrometry. The identities from a local supermarket. Tris buxered saline (TBS) and phosphateof detected allergens were conxrmed by comparing the MS results buxered saline (PBS) tablets were purchased from Amresco, USA.with other species available in GenBank. Selected allergens were The photosensitive xlms were purchased from GE Healthcare,then purixed and sequenced to prove the validity of the strategy for USA. The developer and xxer were purchased from Kodak, USA.allergen identixcation. RapiGest SF surfactant was purchased from Waters Corporation TM, AK, and SCBP were determined to be the most signixcant (Milford, MA, USA), and 37 mm polytetraxuoroethylene (PTFE)allergens and thus were purixed and sequenced. The signature xlters for air sampling were purchased from SKC, Inc. (Eighty Four,peptides for each allergen were assigned and determined to PA, USA). Peptide standards in both light and heavy forms weredevelop an absolute quantixcation (AQUA) MS approach. purchased from GeneMed Synthesis (San Francisco, CA, USA) asThe method reliability was estimated on real samples that were detailed in Table 1.collected from a shrimp processing workplace, where allergenlevels were noticeably high in the main processing station. Northern Shrimp Extracts■ MATERIALS AND METHODS Fresh northern shrimp were collected from a xshing boat in St. John’s−NL Canada. After shell removal, the meat was rinsed with water and stored in liquid nitrogen. Five grams of shrimpChemicals and Materials was homogenized with 50 mL of buxer A (1 M KCl, 25 mM Tris-HCl, pH 8.0, 0.25 M DTT, and 0.5 mM EDTA) and leftAll chemicals were used without further purixcation. Ammonium stirring overnight at 4 °C. The slurry was then centrifuged atsulfate, acetonitrile (ACN), hydrochloric acid, and methanol were 10 000 rpm for 30 min at 4 °C.supplied by ACP (Montreal, Canada). Trypsin sequencing grade The total protein concentration was determined using theenzymes were purchased from Promega (WI, USA). Tris- Bradford assay. The crude extract was used for further characteizing(hydroxymethyl) aminomethane (Tris), dithiotheritol (DTT), 648 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  3. 3. Journal of Proteome Research ArticleTable 1. Standard Material Specixcations and the Multiple Reaction Monitoring (MRM) Transitions of Northern ShrimpTropomyosin (TM) and Arginine Kinase (AK) Signature Peptides in Heavy and Light Forms peptide sequence peptide code purity % average mass Q1(z) m/z Q3(ion) m/z SEEEVFGLQK TM 98.25 1165.8 583 (+2) 217 (b9) SEEE(d8-V)FGLQK d8-TM 98.25 1173.7 587 (+2) 217 (b9) QQLVDDHFLFVSGDR AK 98.27 1776.1 592 (+3) 586 ([M−H2O+3H]+3) QQL(d8-V)DDHFLF(d 8-V)SGDR d16-AK 98.34 1791.1 598 (+3) 592 ([M−H2O+3H]+3)the major allergens. Tropomyosin and AK were targeted for further FA/0.01% TFA/2% ACN and (B) 0.08% FA/0.008% TFA/analysis by purifying them from the crude extracts via protocols 98% ACN. A gradient of 0% B for 10 min, 0−60% B fordeveloped by Helleur et al.20−22 and Garcia-Orozco et al.23 55 min, 60−90% for 3 min, and 90% B for 5 min was applied.Immunoblotting Including a regeneration step, one run was 106 min long. The ESI−MS spectra of the LC-eluting peptides were measured withSera from patients with shrimp allergies were used to demonstrate the same hybrid QqToF-MS/MS system equipped with athe allergenicity of both the shrimp crude extract and purixed nanoelectrospray source (Protana XYZ manipulator). Theallergens. Patients were selected for this study based on clinical nanoelectrospray was generated from a PicoTip needle (10 μmreactivity to shellxsh. In addition, normal sera were used in this study i.d., New Objectives, Wobum, MA, USA) at a voltage of 2400 V.as a negative control. Ethics approval for this study was acquired at The samples were further analyzed by CID−MS/MS, andMonash University as part of an ongoing survey. IgE antibody the resulting spectra were searched against the National Centerimmunoblotting was performed as described previously. 20Briexy, for Biotechnology Information nonredundant (NCBInr) data-proteins were separated using SDS-PAGE and transferred onto a base using a Matrix Science (Mascot) search engine (precursorPVDF membrane.20 After blocking, the membranes were incubated and product ion mass tolerance set at 0.2 Da). Methioninewith patient serum (diluted 1:10 in 1% skimmed milk in PBS-T) oxidation was allowed as a variable modixcation and guanidinylovernight at 4 °C. The membrane was subsequently exposed to (K) as a xxed modixcation when the guanidation derivatizationrabbit polyclonal antihuman IgE antibody (DAKO, USA) and was performed. Peptides were considered identixed if thegoat antirabbit polyclonal antibody labeled with HRP (Promega, Mascot score was over a 95% conxdence limit.USA) with washings between each incubation. Finally, themembranes were incubated with the chemiluminescent substrateand analyzed for IgE reactivity using the ECL technique. 22,24 Air Sample Collection Air samples were collected from a northern shrimp plant duringEnzymatic Digestion the xshing season of 2011. The air samplers, Leland LegacyThe IgE antibody reactive protein bands were excised Sample Pump (SKC, Rochester, MN, USA), were deployed indestained, and the protein trypsin was digested using a standard the peeling, cooking, and packing stations in addition to severalprotocol.20 The tryptic peptides were extracted from the gel xeld blank samples collected outside the plants. The samplersand desalted using C18 ZipTip for MALDI-QqToF analyses. were programmed to collect particulates for an 8-h working The purixed proteins were exposed to several in-solution shift, where the personal breathing zone (PBZ) air samplesenzymatic digestions, to increase the sequence coverage; trypsin, were collected on PTFE xlters at xow rates ranging from 2 toGlu-C V8, or ASP-N enzymes were used in the presence of 3 L min−1. The xow rate of each sampler was calibrated beforeRapiGest surfactant. Trypsin and Glu-C V8 enzymes were incubated and after collection using a Defender 510 air sampling pumpin 50 mM ABC overnight at 37 °C; however, the ASP-N enzymes calibrator (Air-Met Scientixc, Victoria, Australia). The xlterswere incubated in a reaction buxer: 50 mM Tris-HCl and 2.5 mM were subsequently shipped on dry ice to the lab and stored atZnSO4, at pH 8 overnight at 37 °C. The in-solution digestion −80 °C until protein extraction.samples were quenched using the equivalent volume of 1% TFA to The proteins were extracted from the PTFE xlters usingdegrade the acid labile surfactant, and then the samples were freeze- 0.05% RapiGest SF in 0.1 M ammonium bicarbonate, pH 7.8,dried and stored at −80 °C before MS analyses. by shaking at 4 °C overnight. The SF was removed by using 1% formic acid, and the proteins were exposed to tryptic digestion as described above. Finally, the peptides were reconstituted inMass Spectrometry Analysis for Allergen Characterization 100 μL of water and analyzed by LC−MS/MS.The enzymatic peptides were analyzed using two dixerent ionsources, MALDI and ESI, to increase the sequence coverage.The MALDI targets were prepared following the double-layer Allergen Quantixcation by Mass Spectrometryprocedure detailed in another work.20The sample plate was The signature peptides of the major shrimp allergens wereanalyzed in a MALDI-MS/MS at low-energy collision (CID)- determined (as described below) and chemically synthesized toQSTAR XL hybrid quadrupole−quadrupole (Qq)/ToF-MS/ develop the following quantixcation method. Separation andMS equipped with an o-MALDI ion source (Applied Biosystems, analysis were conducted using a Waters Alliance 2795 HPLC systemFoster City, CA, USA). 21 coupled to a Micromass Quattro Ultima (Water Corporation, Peptide separation was conducted using a DIONEX Milford, MA, USA) LC-MSMS operated in electrospray positive-UltiMate3000 Nano LC System (Germering, Germany). A ionization (ESI) mode and adjusted to separate the target250 fmol enzymatic peptide sample was loaded onto a peptides. The peptides were separated on a reversed-phaseprecolumn (300 μm i.d. × 5 mm, C18 PepMap100, 5 μm chromatography column (Kinetex C18, 2.1 mm × 100 mm, 2.6(LCPacking, Sunnyvale, CA)) for desalting and concentrating. μM particle size, Phenomenex, CA, USA) at 20 °C. A gradientPeptides were then separated on a nanoxow analytical column elution was performed, where the aqueous mobile phase (A)(75 μm i.d. × 15 cm, C18 PepMap 100, 3 μm, 100 A (LC consisted of HPLC-grade water with 0.1% formic acid and thePacking, Sunnyvale, CA)) at 180 nL/min using the following organic phase (B) consisted of ACN with 0.1% formic acid.gradient. The aqueous mobile phases consisted of (A) 0.1% The gradient started at 5% B for 0.3 min, 5−90% B for 6 min, 649 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  4. 4. Journal of Proteome Research Articlethen 90% B for 3 min after which it reverted back to the 5% for data to the MASCOT search engine. Due to the lack of DNA0.5 min (total: 11 min run time). A 20 μL injection was used at information in the GeneBank, a phylogenetic tree was developednormal draw speed with a programmed washing procedure. The based on a known protein sequence derived from cDNA, andeluted peptides were desolvated during ESI with a gas xow rate of then the available species in the databases were used (Figure S1,400 L h−1 and a temperature of 250 °C. The ions were accelerated Supporting Information). The protein identity of each band wasthrough the capillary and orixce cone at 3.02 kV and 40 V, reported from the equivalent sequence of the closest species inrespectively. The precursor ions were fragmented using low-energy the database.CID with argon gas and collision energy of 13 eV. The precursorions of the unlabeled and labeled forms of the signature peptides Tropomyosin Purixcation and Sequencingare reported in Table 1. Data processing was performed with Mass The major allergen in shrimp, TM, was specixcally targeted forLynx 4.1 software. Each MS data point given in calibration curves purixcation using multiple precipitation steps and then introducedand sample analysis represents triplicate analyses by LC−MS/MS to a recently developed protocol for primary structure determi-(MRM). Points are a measure of the peak area ratio of selected nation.19,20 This protocol is based on using multiple enzymaticdaughter ions of both the unlabeled and labeled peptide. digestions, dixerent MS ion sources, and a derivatization reaction■ to sequence the global protein with 100% amino acid coverage as shown in Figure 3. Three enzymes were used, trypsin, Gul-C V8, RESULTS and ASP-N, to increase the diversity of the produced peptidesProtein Identixcation and Allergenicity Evaluation using two MS ion sources, ESI and MALDI. The full amino acid sequence of TM was submitted to the UniProtKB/Swiss-ProtInitial experiments were performed on shrimp meat, which was database with accession number P86704.1.isolated from freshly caught shrimp previously snap frozen inliquid nitrogen to quench any protease activities and thenstored at −80 °C. A shrimp crude extract was collected after Arginine Kinase and Sarcoplasmic Ca-Binding Proteinovernight stirring in a suitable buxer at 4 °C, and then the Purixcation and Sequencingproteins were proxled by SDS-PAGE. The allergenicity of each Arginine kinase and SCBP were reported in several species asprotein was examined using nine dixerent sensitized patients’ allergens,13,16,22,23,28 including northern shrimp. These proteinssera. Immunoblotting of the crude extract (Figure 2) shows the were semipurixed together because their isoelectric focusing values are fairly close to each other.23 In the present study, the purixcation steps were monitored by SDS-PAGE as shown in Figure 4A, and the reactivity of the two proteins was examined using a pool of allergenic patient’s sera. The immunoblot of the SCBP shows a double band (Figure 4B) due to having several isoforms that were conxrmed later by MS. Ultimately, the amino acid sequence coverage was 70% and 45% for SCBP and AK, respectively. Their amino acid sequence is reported in Figure 4C and D, where the sequence information was combined from several experimental approaches as described above. Absolute Quantixcation Method Development For quantitative analysis of targeted airborne allergens, an isotopic dilution tandem MS method was developed for TM and AK, where their signature peptides were assigned from their protein sequence data. The criteria for selecting signature peptides were discussed elsewhere.29,30 Accordingly, the best peptide with the highest score of identity was reported for TM and AK in northern shrimp as SEEEVFGLQK and QQLVDDHFLFVSGDR, respectively. The signature peptides were chemically synthesized, in both light and heavy forms, forFigure 2. Clinical reactivity of nine shellxsh allergic patients to developing the proper MRM transitions of the triple quadru-northern shrimp (Pandalus borealis) crude extract using IgE pole mass spectrometer (Table 1). Representative product ionimmunoblotting. The highlighted bands with boxes were labeled spectra for both peptides are shown in Figure 5, where thefrom 1 to 8, which are further analyzed by detailed proteomics. major peptide fragment ions are shown for amino acid sequencing and conxrming the identities of each peptide. Aqueous solutions of the signature peptides were used tovarious reactivity of each protein band with dixerent patients. optimize the LC−MS parameters which enhance the product ionFor instance, the 35 kDa band, a major allergenic protein, signals for better sensitivity. Accordingly, the signature peptideshowed reactivity reached 100% (9/9) with dixerent axnity mixture was chromatographed by a gradient reversed-phaseresponses among patients. Expectedly, normal control sera mode to reach a limit of detection as low as 0.25 nM with linearwere used for immunoblotting and did not show any binding calibration curves ranging from 1 to 1000 nM (Figure 6B). The(data not shown). reliability of the targeted quantixcation method was examined by The reactivity of each band with the patients’ sera was using shrimp crude extract solutions. These samples were trypticreported in Table 2 as well as its identity which was achieved by digested as described in the method section and analyzed in thispeptide mass xngerprinting (PMF). The equivalent band of method. Representative chromatograms for a real extractedeach one that reacted was excised and tryptic digested and then sample are shown in Figure 6A, where each transition representsanalyzed by MALDI QToF mass spectrometry. The massspectral interpretation of each band was performed by uploading 650 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  5. 5. Journal of Proteome Research ArticleTable 2. List of VAPEEHPVLLWEAPLNPKthe Reported SYELPDGQVITISNERNorth Shrimp DITNYLGK isoforms SYELPDGQVITIGNERAllergens That GYSFTTTAERHave Been EITGLAPSSIKIdentixed EEYDESGPGIVHRKUsing peptide EITALAPSSIK N/A N/A sequencing SYELPDGQVITISNERAVFPSIVGREGYSFTTTAEREEYDESGPGIVHRGIDGFGR Figure 4 (C)Proteomics N/A Figure 3Mass HVYNEMKPENIPWSKGAGQNIIPSSTGAAKAGAHMKGGAKAGAEYIVESTGVFTTIEKAGAHMKGGAKLTGMAFSpectrometry RVPTPDVSVVDLTVRAGIQLSKLTQEAVADLER N/Ain Crude ELQARIEELLDEAGGATSAQIELNKDEAGGATSAQIELNKKRDLKLTQEAVDLLRQLEEAELQARIEELELSQVRQ DYEINELNIQVNDLRDKKKLFExtract after EILTQEAVADLERQIEEAEEIAALNLAKLADELRAEQEHAQTQEK EGGWFLIEEDEEALKTELRDEHaving Them regular Figure 4 (D) EALKTELRGLDPEALTGKHPP KEGFQLMDREvaluatedagainstPatientsSera accession # gi|220172365gi| gi|125995161 gi|68272073gi| gi|242006231 gi|226693231 gi|238477327 gi|152013721 207298829gi|3907622 gi|136223 229256 number of peptides 22 10 9 11 14 9 5 1 sequence coverage % 6.7 27.8 100 49 32 45 70 4 MASCOT score 1004 214 123 98 62 544 76 51 size 33 31 37 227 40 22 23 19 (kDa) Reactivity = reactivity % number of subjects that 100 22 44 11 44 33 33 44 react with the band/total a number of subjects. Pandalus borealis northern shrimp (Pan b1) Litopenaeus Crangon crangon (North Sea Litopenaeus ref 22 vannamei (pacific shrimp) (Cra c2) vannamei (White allergen code white shrimp); Litopenaeus vannamei (White shrimp) (Lit v3) ref 27 shrimp) (Lit v4) Crangon crangon (North Sea shrimp) (Cra c6) refs 25, 26 Sarcoplasmic Glyceraldehyde- calcium-binding 3-phosphate protein protein name dehydrogenase Troponin C Myosin heavy Myosin light chain Arginine chain Tropomyosin kinase Actin band 5 and 1 number 4 5 8 5 3 3 2 a 651 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  6. 6. Journal of Proteome Research ArticleFigure 3. Full amino acid sequence of northern shrimp TM usingcomprehensive mass spectrometry. The dixerent underlined proteinregions are derived from dixerent proteases and/or MS ionizationapproaches. The chosen signature peptide is indicated by the blue box.a signal for a specixc signature peptide or one of their internalstandards.Shrimp Workplace Sampling and AnalysisDuring the shrimp xshing season in the summer of 2011, aprocessing plant located on the northern shore of the Province ofQuebec, Canada, was targeted to be a model for our approach.Personal breathing zone air samples were collected using PTFExlters attached to air pumps. The air sample collection for theallergen proxle was recently standardized and has been followed inthis study.29,30 The samplers were deployed on workers toiling incooking, peeling, and packing areas for 8 h of operation and shrimpprocessing. The target allergens were extracted from the xlters usinga standard protocol, and the eluted allergens were tryptic digestedand then analyzed by LC−MS/MS.29,30 The levels of both targetedallergens (TM and AK) were reported in Figure 7.■ DISCUSSIONAllergenomics is a subxeld of proteomics where the reactiveproteins, along with human sera’s IgE, are screened andtargeted for further characterization (Figure 1). Recent studieson the global burden of disease indicate that occupational lungdiseases are caused by exposure to airborne agents such asallergens. In addition, it is suggested that up to 15% of adultasthma is attributed to occupational exposure. 31 While occupational respiratory diseases are still largely under-recognized, they remain poorly diagnosed and managed. 32−35Various epidemiological studies among seafood processorsindicate that the prevalence of occupational asthma is between2% and 36%, while it is more commonly associated with Figure 4. (A) Northern shrimp arginine kinase (AK) and sarcoplasmicshellxsh processing.19 The challenge for bioaerosol exposure Ca-binding (SCBP) proteins purixcation steps, where crude extractassessment is the lack of methodological advancements in the (CE), 70% supernatant (70% S), 70% pellet (70% P), 90% supernatantaccurate and sensitive quantixcation of biomarker exposure. 33 (90% S), and 90% pellet (90% P) after ammonium sulfateIn the present study a novel method was developed to detect precipitation. (B) An immunoblot of both proteins against a pool ofand quantify the most potent allergenic proteins from northern patients’ sera IgE, where the double bands represent the isoforms. (C)shrimp in air samples from shrimp processing workplaces. The amino acid sequence of SCBP and the detected isoform peptides. (D) The amino acid sequence of AK and the detected isoform Several allergenic proteins are known to be unstable under peptides. Note: In (C) and (D), red: covered experimentally, black:heat and protease conditions. AK is one of these allergens covered by similarity, and blue: isoform motifs.which entails working with fresh meat stored at −80 °C aftersnap freezing them in liquid nitrogen. For this study, thepatients were recruited based on their clinical history ofreactivity to shrimp. Total IgE and shrimp-specixc IgE in the patient serum were quantixed using the ImmunoCAP system (Thermo Scientixc). Total IgE ranged from 56 to 3401 kU/L, and shrimp-specixc IgE ranged from 0.5 to 6.65 kU/L. 652 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  7. 7. Journal of Proteome Research ArticleFigure 5. Product ion mass spectra of northern shrimp: (A) tropomyosins signature peptide [SEEEVFGLQK] and (B) arginine kinase’s signaturepeptide (AK) [QQLVDDHFLFVSGDR].The shrimp crude extract was successfully proxled in SDS- Tropomyosin and AK were both detected by MS in band 5.PAGE, which was enough to study its allergenicity against These proteins were purixed, and their allergenicity waspatients’ sera. As shown in Figure 2, a couple of bands (5 and 7) conxrmed in separated forms. Tropomyosin is precipitatedshow reactivity with all patients’ sera, and therefore the contents of out at 70% saturation by ammonium sulfate, while on the otherthese bands are major allergen(s). Tropomyosin is a major hand the AK precipitated at 90% saturation as seen in Figureallergen in dixerent seafood species, and its α-helix primary 4A. A complete amino acid sequence of TM was covered bystructure is also known to develop hydrophobic interactions to MSMS using several proteases and ion sources. The TMform dimers and under certain conditions higher oligomers. 20 The structure makes it very susceptible to the proteases and veryidentity of these two bands was identixed using MS, revealing they excient in ionization.40 In contrast, AK and SCBP, globularare both related to TM. Normal control sera were used for proteins, show resistance to both Glu-C V8 and ASP-Nimmunoblotting and did not show any binding (data not shown) digestion even after using RapiGest. The digestion was very Signixcant bands were also targeted for protein identixcation poor in the case of AK but relatively excient in the case of TMusing MS, and the results are summarized in Table 2. The and SCBP, which rexects the resulting sequence coverage.reactivity of each band was calculated as the percentage of allergic Tropomyosin has a highly conserved primary structure andto nonallergic patients. Most allergens exist in several variants shares a high amino acid sequence identity among crustaceans.(isoallergens), which are recognized dixerently by patient IgE, as The amino acid sequence identity of tropomyosin among eightshown in Figure 4A, where the allergenicity of SCBP against the dixerent shrimp species ranges from 96% to 100%. In this studypool of patient sera shows double bands.27,36−38 Noticeably, only the major allergens, TM and AK, were targeted forprotein isoforms were detected in some of these allergens, which quantixcation. The signature peptides for both allergens wereare either related to dixerent gene contributions in expression or selected and evaluated based on selection criteria of signaturedue to alternative splicing of the same unique gene. 35,39 Table 2 peptides and examined by an NCBI protein blast testhighlights the sites of heterogeneity for each isoallergen and algorithm.29,30 The method was developed to maintain the lowestreports their peptide sequences. limit of detection to increase the sensitivity of routine analysis for 653 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  8. 8. Journal of Proteome Research ArticleFigure 6. Representative real sample chromatograms for the signature peptides of northern shrimp tropomyosin (TM) and arginine kinase (AK)along with their labeled forms d8-TM and d16-AK (A), respectively. Representative calibration curves for TM and AK (B), where the response(y-axis) is the area ratio of the signature peptide and its internal standard. reduce his or her exposure to the aeroallergens because the proteins are spread all over the plant. More data are needed to be able to draw a trend and propose preventative actions to avoid occupational illness. The level of AK everywhere inside the plant was slightly higher than TM, although the natural abundance of TM in shrimp tissue is higher. This dixerence between AK and TM was also observed and discussed in previous studies in crab plants, where most of the AK comes from the hemolymph which is easily aerosolized or steamed in cleaning and cooking areas. 29,30 A bigger study is being conducted in Quebec-Canada following this approach, which we believe will allow us to propose curative actions and help us to rationalize allergen exposure in dixerent workstations.Figure 7. Concentration of tropomyosin and arginine kinase in airsamples from three dixerent workstations in a northern shrimpprocessing plant in the Province of Quebec. Number of samples in the ■ S ASSOCIATED CONTENTpeeling station n = 4 and in the cooking and packing station n = 1each. * Supporting Information Figure S1. Phylogenetic tree based on the tropomyosin amino acid sequences for various crustacean species (accession number) and compared to human and chicken tropomyosin American lobsterscreening workplaces. The method was very selective, reprodu- (O44119); northern shrimp (P86704); Kuruma prawncible, and accurate for measuring the level of these allergens in air (AB270630.1); Black tiger prawn (HM486525.1); Snow crab (A2samples (Figure 6). V735); Horsehair crab (BAF47269); Cockroach (AAD19606); In terms of processing shrimp, the cooking and peeling steps Locust (P31816); Dust mite (AAB69424); Storage mite (AAQ-involve removing the shell from the cooked shrimp. The packing 54614); Pacixc oyster (AAK96889); Blue mussel (AAA82259);step readies the cooked shrimp for consumption in an appropriate Human (AAB59509); Chicken (AAA49112). This material ispackage for shipment. These processing steps were judged as available free of charge via the Internet at http://pubs.acs.org.being the most relevant to conduct air monitoring. The airsamples were collected during 8 h of operation and processing. Jeebhay and Cartier (2010) surveyed several studies andfound ranges for the total inhalable airborne particulate(0.001−11.293 mg/m3), total protein (0.001−6.4 mg/m3), ■ AUTHOR INFORMATION Corresponding Authorand allergens (0.001−75.748 μg/m 3).33 This study involved adeeper exploration and more specixcs by reporting the mean levels *Phone: (+1) 416-586-4800. Ext 8268. Fax: (+1) 416-586-of indoor aerosolized allergens TM and AK: 125 and 480 ng/m 3, 4200. E-mail: abdel@lunenfeld.ca.respectively. On the basis of these results, it could be challengingto relocate a sensitized worker somewhere else in the plant to Notes The authors declare no competing xnancial interest. 654 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  9. 9. Journal of Proteome Research Article■ ACKNOWLEDGMENTSThis research was partially funded by the National Sciences and of the black tiger shrimp (penaeus monodon). Int. Arch. Allergy Immunol. 2008, 146 (2), 91−98. (17) Ayuso, R.; Grishina, G.; Bardina, L.; Carrillo, T.; Blanco, C.;Engineering Research Council (NSERC) and by the Australian Ibáñez, M.; Sampson, H.; Beyer, K. Myosin light chain is a novelResearch Council (ARC)- Future Fellowship Award (Dr. Andreas shrimp allergen, lit v 3. J. Allergy Clin. Immunol. 2008, 122 (4), 795−Lopata). We would like to acknowledge Memorial University of 802.Newfoundland (MUN) and the Department of Chemistry and (18) Ayuso, R.; Sánchez-Garcia, S.; Lin, J.; Fu, Z.; Ibáñez, M.;IRSST for xnancial support. The clinical biochemistry lab in Carrillo, T.; Blanco, C.; Goldis, M.; Bardina, L.; Sastre, J.; Sampson, H.Eastern Health (Dr. Edward Randell) and MUN Genomic and A. Greater epitope recognition of shrimp allergens by children than byProteomics facility are highly acknowledged for giving access to adults suggests that shrimp sensitization decreases with age. J. Allergythe mass spectrometry machines. Finally, the authors acknowl- Clin. Immunol. 2010, 125 (6), 1286−1293. (19) Abdel Rahman, A. M.; Helleur, R. J.; Jeebhay, M. F.; Lopata, A.edged Prof. Robyn O’Hehir (The Alfred Hospital, Melbourne, L. Characterization of Seafood Proteins Causing Allergic Diseases,VIC, Australia) for supplying patient sera. Allergic Diseases. Highlights in the Clinic, Mechanisms and Treatment; Prof. Celso Pereira, Ed.; InTech, 2012, ISBN: 978-953-51-0227-4.■ Available from: http://www.intechopen.com/books/allergic-diseases- highlights-in-the-clinic-mechanisms-and-treatment/characterization-of- REFERENCES seafood-proteins-causing-allergic-diseases. (1) Sampson, H. A. Fatal food-induced anaphylaxis. Allergy 1998, 53, (20) Abdel Rahman, A. M.; Lopata, A. L.; O’Hehir, R. 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  10. 10. Journal of Proteome Research Article (31) American Thoracic Society.. Occupational contribution to theburden of airway disease. Am. J. Respir. Crit. Care Med. 2003, 167,787−797. (32) Jeebhay, M. F; Quirce, S. Occupational asthma in thedeveloping and industrialized world: a review. Int. J. Tuberc. LungDis. 2007, 11 (2), 122−33. (33) Jeebhay, M. F.; Cartier, A. Seafood workers and respiratorydisease: an update. Curr. Opin. Allergy Clin. Immunol. 2010, 10 (2),104−13 Review. (34) Jeebhay, M. F.; Robins, T. G.; Miller, M. E.; Bateman, E.; Smuts,M.; Baatjies, R.; Lopata, A. L. Occupational allergy and asthma amongsalt water fish processing workers. Am. J. Ind. Med. 2008, 51 (12),899−910. (35) Jeebhay, M. F.; Robins, T. G.; Seixas, N.; Baatjies, R.; George, D.A.; Rusford, E.; Lehrer, S. B.; Lopata, A. L. Environmental exposurecharacterization of fish processing workers. Ann. Occup. Hyg. 2005, 49(5), 423−37. (36) Christensen, L,H; Riise, E.; Bang, L.; Zhang, C.; Lund, K.Isoallergen variations contribute to the overall complexity of effectorcell degranulation: effect mediated through differentiated IgE affinity.J. Immunol. 2010, 184 (9), 4966−4972. (37) Barre, L.; Fournel-Gigleux, S.; Finel, M.; Netter, P.; Magdalou,J.; Ouzzine, M. Substrate specificity of the human UDP-glucuronosyl-transferase UGT2B4 and UGT2B7. Identification of a critical aromaticamino acid residue at position 33. FEBS J. 2007, 274 (5), 1256−1264. ̈ ̈ (38) Seppala, U.; Dauly, C.; Robinson, S.; Hornshaw, M.; Larsen, J.N.; Ipsen, H. Absolute Quantification of Allergens from ComplexMixtures: A New Sensitive Tool for Standardization of AllergenExtracts for Specific Immunotherapy. J. Proteome Res. 2011, 10 (4),2113−2122. (39) Eduard, W.; Heederik, D.; Duchaine, C.; Green, B. J. Bioaerosolexposure assessment in the workplace: the past, present and recentadvances. J. Environ. Monit. 2012, 14 (2), 334−339. (40) Vijay-Kumar, S.; Cook, W. J. Structure of a sarcoplasmiccalcium-binding protein from Nereis diversicolor refined at 2·0 Åresolution. J. Mol. Biol. 1994, 224 (2), 413−426. 656 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  11. 11. Journal of Proteome Research Article (31) American Thoracic Society.. Occupational contribution to theburden of airway disease. Am. J. Respir. Crit. Care Med. 2003, 167,787−797. (32) Jeebhay, M. F; Quirce, S. Occupational asthma in thedeveloping and industrialized world: a review. Int. J. Tuberc. LungDis. 2007, 11 (2), 122−33. (33) Jeebhay, M. F.; Cartier, A. Seafood workers and respiratorydisease: an update. Curr. Opin. Allergy Clin. Immunol. 2010, 10 (2),104−13 Review. (34) Jeebhay, M. F.; Robins, T. G.; Miller, M. E.; Bateman, E.; Smuts,M.; Baatjies, R.; Lopata, A. L. Occupational allergy and asthma amongsalt water fish processing workers. Am. J. Ind. Med. 2008, 51 (12),899−910. (35) Jeebhay, M. F.; Robins, T. G.; Seixas, N.; Baatjies, R.; George, D.A.; Rusford, E.; Lehrer, S. B.; Lopata, A. L. Environmental exposurecharacterization of fish processing workers. Ann. Occup. Hyg. 2005, 49(5), 423−37. (36) Christensen, L,H; Riise, E.; Bang, L.; Zhang, C.; Lund, K.Isoallergen variations contribute to the overall complexity of effectorcell degranulation: effect mediated through differentiated IgE affinity.J. Immunol. 2010, 184 (9), 4966−4972. (37) Barre, L.; Fournel-Gigleux, S.; Finel, M.; Netter, P.; Magdalou,J.; Ouzzine, M. Substrate specificity of the human UDP-glucuronosyl-transferase UGT2B4 and UGT2B7. Identification of a critical aromaticamino acid residue at position 33. FEBS J. 2007, 274 (5), 1256−1264. ̈ ̈ (38) Seppala, U.; Dauly, C.; Robinson, S.; Hornshaw, M.; Larsen, J.N.; Ipsen, H. Absolute Quantification of Allergens from ComplexMixtures: A New Sensitive Tool for Standardization of AllergenExtracts for Specific Immunotherapy. J. Proteome Res. 2011, 10 (4),2113−2122. (39) Eduard, W.; Heederik, D.; Duchaine, C.; Green, B. J. Bioaerosolexposure assessment in the workplace: the past, present and recentadvances. J. Environ. Monit. 2012, 14 (2), 334−339. (40) Vijay-Kumar, S.; Cook, W. J. Structure of a sarcoplasmiccalcium-binding protein from Nereis diversicolor refined at 2·0 Åresolution. J. Mol. Biol. 1994, 224 (2), 413−426. 656 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  12. 12. Journal of Proteome Research Article (31) American Thoracic Society.. Occupational contribution to theburden of airway disease. Am. J. Respir. Crit. Care Med. 2003, 167,787−797. (32) Jeebhay, M. F; Quirce, S. Occupational asthma in thedeveloping and industrialized world: a review. Int. J. Tuberc. LungDis. 2007, 11 (2), 122−33. (33) Jeebhay, M. F.; Cartier, A. Seafood workers and respiratorydisease: an update. Curr. Opin. Allergy Clin. Immunol. 2010, 10 (2),104−13 Review. (34) Jeebhay, M. F.; Robins, T. G.; Miller, M. E.; Bateman, E.; Smuts,M.; Baatjies, R.; Lopata, A. L. Occupational allergy and asthma amongsalt water fish processing workers. Am. J. Ind. Med. 2008, 51 (12),899−910. (35) Jeebhay, M. F.; Robins, T. G.; Seixas, N.; Baatjies, R.; George, D.A.; Rusford, E.; Lehrer, S. B.; Lopata, A. L. Environmental exposurecharacterization of fish processing workers. Ann. Occup. Hyg. 2005, 49(5), 423−37. (36) Christensen, L,H; Riise, E.; Bang, L.; Zhang, C.; Lund, K.Isoallergen variations contribute to the overall complexity of effectorcell degranulation: effect mediated through differentiated IgE affinity.J. Immunol. 2010, 184 (9), 4966−4972. (37) Barre, L.; Fournel-Gigleux, S.; Finel, M.; Netter, P.; Magdalou,J.; Ouzzine, M. Substrate specificity of the human UDP-glucuronosyl-transferase UGT2B4 and UGT2B7. Identification of a critical aromaticamino acid residue at position 33. FEBS J. 2007, 274 (5), 1256−1264. ̈ ̈ (38) Seppala, U.; Dauly, C.; Robinson, S.; Hornshaw, M.; Larsen, J.N.; Ipsen, H. Absolute Quantification of Allergens from ComplexMixtures: A New Sensitive Tool for Standardization of AllergenExtracts for Specific Immunotherapy. J. Proteome Res. 2011, 10 (4),2113−2122. (39) Eduard, W.; Heederik, D.; Duchaine, C.; Green, B. J. Bioaerosolexposure assessment in the workplace: the past, present and recentadvances. J. Environ. Monit. 2012, 14 (2), 334−339. (40) Vijay-Kumar, S.; Cook, W. J. Structure of a sarcoplasmiccalcium-binding protein from Nereis diversicolor refined at 2·0 Åresolution. J. Mol. Biol. 1994, 224 (2), 413−426. 656 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656
  13. 13. Journal of Proteome Research Article (31) American Thoracic Society.. Occupational contribution to theburden of airway disease. Am. J. Respir. Crit. Care Med. 2003, 167,787−797. (32) Jeebhay, M. F; Quirce, S. Occupational asthma in thedeveloping and industrialized world: a review. Int. J. Tuberc. LungDis. 2007, 11 (2), 122−33. (33) Jeebhay, M. F.; Cartier, A. Seafood workers and respiratorydisease: an update. Curr. Opin. Allergy Clin. Immunol. 2010, 10 (2),104−13 Review. (34) Jeebhay, M. F.; Robins, T. G.; Miller, M. E.; Bateman, E.; Smuts,M.; Baatjies, R.; Lopata, A. L. Occupational allergy and asthma amongsalt water fish processing workers. Am. J. Ind. Med. 2008, 51 (12),899−910. (35) Jeebhay, M. F.; Robins, T. G.; Seixas, N.; Baatjies, R.; George, D.A.; Rusford, E.; Lehrer, S. B.; Lopata, A. L. Environmental exposurecharacterization of fish processing workers. Ann. Occup. Hyg. 2005, 49(5), 423−37. (36) Christensen, L,H; Riise, E.; Bang, L.; Zhang, C.; Lund, K.Isoallergen variations contribute to the overall complexity of effectorcell degranulation: effect mediated through differentiated IgE affinity.J. Immunol. 2010, 184 (9), 4966−4972. (37) Barre, L.; Fournel-Gigleux, S.; Finel, M.; Netter, P.; Magdalou,J.; Ouzzine, M. Substrate specificity of the human UDP-glucuronosyl-transferase UGT2B4 and UGT2B7. Identification of a critical aromaticamino acid residue at position 33. FEBS J. 2007, 274 (5), 1256−1264. ̈ ̈ (38) Seppala, U.; Dauly, C.; Robinson, S.; Hornshaw, M.; Larsen, J.N.; Ipsen, H. Absolute Quantification of Allergens from ComplexMixtures: A New Sensitive Tool for Standardization of AllergenExtracts for Specific Immunotherapy. J. Proteome Res. 2011, 10 (4),2113−2122. (39) Eduard, W.; Heederik, D.; Duchaine, C.; Green, B. J. Bioaerosolexposure assessment in the workplace: the past, present and recentadvances. J. Environ. Monit. 2012, 14 (2), 334−339. (40) Vijay-Kumar, S.; Cook, W. J. Structure of a sarcoplasmiccalcium-binding protein from Nereis diversicolor refined at 2·0 Åresolution. J. Mol. Biol. 1994, 224 (2), 413−426. 656 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656

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