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Comprehensive Proteomics Approach in Characterizing andQuantifying Allergenic Proteins from Northern Shrimp: TowardBetter Occupational Asthma PreventionAnas M. Abdel Rahman,*,†,‡ Sandip D. Kamath,§ Sebastien Gagne,∥ 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 Cook University, Australia∥

Institut de Recherche Robert-Sauve en Sante et Securite au Travail, Montreal, Quebec, CanadaS

* Supporting Information

ABSTRACT: Occupational asthma is a major chronic healthdilemma among workers involved in the seafood industry. Severalproteins notoriously known to cause asthma have been reported indifferent 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 profiled by gelelectrophoresis. Allergenic proteins were identified based on theirreactivity to patient sera and were structurally identified usingtandem mass spectrometry. Northern shrimp tropomyosin, argininekinase, and sarcoplasmic calcium-binding protein were found to bethe most significant allergens. Multiple proteolytic enzymes enabled100% coverage of the sequence of shrimp tropomyosin by tandemmass specrometry. Only partial sequence coverage was obtained,however, for the shrimp allergen arginine kinase. Signature peptides,for both tropomyosin and arginine kinase, were assigned andsynthesized for use in developing the multiple reaction monitoringtandem mass spectrometric method. Subsequently, air samples werecollected from a shrimp processing plant and two aerosolized proteins quantified using tandem mass specrometry. Allergens were detectedin all areas of the plant, reaching levels as high as 375 and 480 ng/m3 for tropomyosine and arginine kinase, respectively. Tropomyosine ismuch more abundant than arginine kinase in shrimp tissues, so the high levels of arginine kinase suggest it is more easily aerosolized. Thepresent 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,which is responsible for hundreds of fatalities annually worldwide.1Shrimp allergy affects about 2% of the general population world-wide.2−4 In recent decades, seafood consumption has dramaticallyincreased as a healthier diet choice, which in turn has increased thenumber of people engaged in the seafood industry. These workersare frequently exposed to aeroallergens that cause type-I hyper-sensitivity. Northern shrimp (Pandalus borealis), caught in theNorth Atlantic and North Pacific oceans, are the most commonlyconsumed shrimp in North America. The lack of molecular dataon allergenic proteins from most seafood species requires thedevelopment of comprehensive strategies to study the biochemicalcharacteristics of these allergens.

© 2012 American Chemical Society

■ Double-blind placebo controlled food challenges (DBPCFCs)were performed to determine threshold shrimp doses forindividuals with shrimp allergies. The threshold doses rangedfrom 14 to 16 g of shrimp equivalent to 32 mg of protein andfrom 5 to 600 mg of protein for different fish.4−7 Seafood allergenquantification was initiated in 1997 by Lehrer’s group using asandwich Enzyme-linked immunosorbent assay (ELISA) approachfor targeting brown shrimp tropomyosin (TM).8 The detectionlimit was 4 ng/mL, and the assay was applied to detect TM indifferent crustacean species such as crab and lobster. Recently, themethod was optimized to evaluate the level of crustacean majorReceived: August 8, 2012Published: December 26, 2012

647 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656

Journal of Proteome Research Article

Figure 1. Allergenomics strategy for allergen discovery using functional proteomics followed by a mass spectrometric approach for molecularcharacterization.17

allergen, TM, in processed food9,10 with a limit of detection ofabout 2.5 mg/kg.11

Although TM is the major crustacean allergen responsible foringestion-related allergic reactions,12 other allergens wereidentified and characterized such as arginine kinase (AK),13,14

sarcoplasmic calcium-binding protein (SCBP),15,16 and myosin-light chain (MLC).17,18

This study introduces a functional proteomics strategy(allergenomic) to evaluate the potential allergenic proteins in north-ern shrimp (NS) as summarized in Figure 1.19 Sera from sensitizedpatients were used to evaluate the proteomics profile of NS whichwas subsequently identified using mass spectrometry. The identitiesof detected allergens were confirmed by comparing the MS resultswith other species available in GenBank. Selected allergens werethen purified and sequenced to prove the validity of the strategy forallergen identification. TM, AK, and SCBP were determined to be the most significantallergens and thus were purified and sequenced. The signaturepeptides for each allergen were assigned and determined todevelop an absolute quantification (AQUA) MS approach.The method reliability was estimated on real samples that werecollected from a shrimp processing workplace, where allergenlevels were noticeably high in the main processing station.

ethylenediaminetetraacetic acid (EDTA), formic acid (FA),ammonium bicarbonate, o-ethylisourea hemisulfate, ammonium-hydroxide, horseradish peroxidase (HRP), chemiluminescentsubstrate, sodium dodecylsulfate (SDS), ammonium formate, andα-cyano-4-hydroxycinamic acid (HCCA) matrix were purchasedfrom Sigma-Aldrich (St. Louis, MO, USA). The Bradford assay kitand PVDF immunoblot membranes were purchased from BioRad(Hercules, CA, USA), and dialysis bags were purchased fromFischer Scientific (Roncho Dominguez, CA, USA). ZipTip C18

filters were purchased for desalting from Millipore Corporation(Bedford, MA, USA). Powdered skimmed milk was purchasedfrom a local supermarket. Tris buffered saline (TBS) and phosphatebuffered saline (PBS) tablets were purchased from Amresco, USA.The photosensitive films were purchased from GE Healthcare,USA. The developer and fixer were purchased from Kodak, USA.RapiGest SF surfactant was purchased from Waters Corporation(Milford, MA, USA), and 37 mm polytetrafluoroethylene (PTFE)filters for air sampling were purchased from SKC, Inc. (Eighty Four,PA, USA). Peptide standards in both light and heavy forms werepurchased from GeneMed Synthesis (San Francisco, CA, USA) asdetailed in Table 1.

Northern Shrimp Extracts

■MATERIALS AND METHODS

Chemicals and Materials

All chemicals were used without further purification. Ammoniumsulfate, acetonitrile (ACN), hydrochloric acid, and methanol weresupplied by ACP (Montreal, Canada). Trypsin sequencing gradeenzymes were purchased from Promega (WI, USA). Tris-(hydroxymethyl) aminomethane (Tris), dithiotheritol (DTT),

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Fresh northern shrimp were collected from a fishing boat in St.John’s−NL Canada. After shell removal, the meat was rinsedwith water and stored in liquid nitrogen. Five grams of shrimpwas homogenized with 50 mL of buffer A (1 M KCl, 25 mMTris-HCl, pH 8.0, 0.25 M DTT, and 0.5 mM EDTA) and leftstirring overnight at 4 °C. The slurry was then centrifuged at10 000 rpm for 30 min at 4 °C. The total protein concentration was determined using theBradford assay. The crude extract was used for further characteizing

dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656


Table 1. Standard Material Specifications and the Multiple Reaction Monitoring (MRM) Transitions of Northern ShrimpTropomyosin (TM) and Arginine Kinase (AK) Signature Peptides in Heavy and Light Forms

peptide sequence

SEEEVFGLQKSEEE(d8-V)FGLQKQQLVDDHFLFVSGDRQQL(d8-V)DDHFLF(d8-V)SGDR

peptide code

TMd8-TMAKd16-AK

purity %

98.2598.2598.2798.34

average mass

1165.81173.71776.11791.1

Q1(z) m/z

583587592598

(+2)(+2)(+3)(+3)

217217586592

Q3(ion) m/z

(b9)(b9)([M−H2O+3H]+3)([M−H2O+3H]+3)

the major allergens. Tropomyosin and AK were targeted for furtheranalysis by purifying them from the crude extracts via protocolsdeveloped by Helleur et al.20−22 and Garcia-Orozco et al.23Immunoblotting

Sera from patients with shrimp allergies were used to demonstratethe allergenicity of both the shrimp crude extract and purifiedallergens. Patients were selected for this study based on clinicalreactivity to shellfish. In addition, normal sera were used in this studyas a negative control. Ethics approval for this study was acquired atMonash University as part of an ongoing survey. IgE antibodyimmunoblotting was performed as described previously.20Briefly,proteins were separated using SDS-PAGE and transferred onto aPVDF membrane.20 After blocking, the membranes were incubatedwith patient serum (diluted 1:10 in 1% skimmed milk in PBS-T)overnight at 4 °C. The membrane was subsequently exposed torabbit polyclonal antihuman IgE antibody (DAKO, USA) andgoat antirabbit polyclonal antibody labeled with HRP (Promega,USA) with washings between each incubation. Finally, themembranes were incubated with the chemiluminescent substrateand analyzed for IgE reactivity using the ECL technique.22,24

Enzymatic Digestion

FA/0.01% TFA/2% ACN and (B) 0.08% FA/0.008% TFA/98% ACN. A gradient of 0% B for 10 min, 0−60% B for55 min, 60−90% for 3 min, and 90% B for 5 min was applied.Including a regeneration step, one run was 106 min long. TheESI−MS spectra of the LC-eluting peptides were measured withthe same hybrid QqToF-MS/MS system equipped with ananoelectrospray source (Protana XYZ manipulator). Thenanoelectrospray was generated from a PicoTip needle (10 μmi.d., New Objectives, Wobum, MA, USA) at a voltage of 2400 V. The samples were further analyzed by CID−MS/MS, andthe resulting spectra were searched against the National Centerfor Biotechnology Information nonredundant (NCBInr) data-base using a Matrix Science (Mascot) search engine (precursorand product ion mass tolerance set at 0.2 Da). Methionineoxidation was allowed as a variable modification and guanidinyl(K) as a fixed modification when the guanidation derivatizationwas performed. Peptides were considered identified if theMascot score was over a 95% confidence limit.

Air Sample Collection

The IgE antibody reactive protein bands were exciseddestained, and the protein trypsin was digested using a standardprotocol.20 The tryptic peptides were extracted from the geland desalted using C18 ZipTip for MALDI-QqToF analyses. The purified proteins were exposed to several in-solutionenzymatic digestions, to increase the sequence coverage; trypsin,Glu-C V8, or ASP-N enzymes were used in the presence ofRapiGest surfactant. Trypsin and Glu-C V8 enzymes were incubatedin 50 mM ABC overnight at 37 °C; however, the ASP-N enzymeswere incubated in a reaction buffer: 50 mM Tris-HCl and 2.5 mMZnSO4, at pH 8 overnight at 37 °C. The in-solution digestionsamples were quenched using the equivalent volume of 1% TFA todegrade the acid labile surfactant, and then the samples were freeze-dried and stored at −80 °C before MS analyses.

Mass Spectrometry Analysis for Allergen Characterization

The enzymatic peptides were analyzed using two different ionsources, MALDI and ESI, to increase the sequence coverage.The MALDI targets were prepared following the double-layerprocedure detailed in another work.20The sample plate wasanalyzed in a MALDI-MS/MS at low-energy collision (CID)-QSTAR XL hybrid quadrupole−quadrupole (Qq)/ToF-MS/MS equipped with an o-MALDI ion source (Applied Biosystems,Foster City, CA, USA).21

Peptide separation was conducted using a DIONEXUltiMate3000 Nano LC System (Germering, Germany). A250 fmol enzymatic peptide sample was loaded onto aprecolumn (300 μm i.d. × 5 mm, C18 PepMap100, 5 μm(LCPacking, Sunnyvale, CA)) for desalting and concentrating.Peptides were then separated on a nanoflow analytical column(75 μm i.d. × 15 cm, C18 PepMap 100, 3 μm, 100 A (LCPacking, Sunnyvale, CA)) at 180 nL/min using the followinggradient. The aqueous mobile phases consisted of (A) 0.1%649

Air samples were collected from a northern shrimp plant duringthe fishing season of 2011. The air samplers, Leland LegacySample Pump (SKC, Rochester, MN, USA), were deployed inthe peeling, cooking, and packing stations in addition to severalfield blank samples collected outside the plants. The samplerswere programmed to collect particulates for an 8-h workingshift, where the personal breathing zone (PBZ) air sampleswere collected on PTFE filters at flow rates ranging from 2 to3 L min−1. The flow rate of each sampler was calibrated beforeand after collection using a Defender 510 air sampling pumpcalibrator (Air-Met Scientific, Victoria, Australia). The filterswere subsequently shipped on dry ice to the lab and stored at−80 °C until protein extraction. The proteins were extracted from the PTFE filters using0.05% RapiGest SF in 0.1 M ammonium bicarbonate, pH 7.8,by shaking at 4 °C overnight. The SF was removed by using 1%formic acid, and the proteins were exposed to tryptic digestionas described above. Finally, the peptides were reconstituted in100 μL of water and analyzed by LC−MS/MS.

Allergen Quantification by Mass Spectrometry

The signature peptides of the major shrimp allergens weredetermined (as described below) and chemically synthesized todevelop the following quantification method. Separation andanalysis were conducted using a Waters Alliance 2795 HPLC systemcoupled to a Micromass Quattro Ultima (Water Corporation,Milford, MA, USA) LC-MSMS operated in electrospray positive-ionization (ESI) mode and adjusted to separate the targetpeptides. The peptides were separated on a reversed-phasechromatography column (Kinetex C18, 2.1 mm × 100 mm, 2.6μM particle size, Phenomenex, CA, USA) at 20 °C. A gradientelution was performed, where the aqueous mobile phase (A)consisted of HPLC-grade water with 0.1% formic acid and theorganic phase (B) consisted of ACN with 0.1% formic acid.The gradient started at 5% B for 0.3 min, 5−90% B for 6 min,


Journal of Proteome Research

then 90% B for 3 min after which it reverted back to the 5% for0.5 min (total: 11 min run time). A 20 μL injection was used atnormal draw speed with a programmed washing procedure. Theeluted peptides were desolvated during ESI with a gas flow rate of400 L h−1 and a temperature of 250 °C. The ions were acceleratedthrough the capillary and orifice cone at 3.02 kV and 40 V,respectively. The precursor ions were fragmented using low-energyCID with argon gas and collision energy of 13 eV. The precursorions of the unlabeled and labeled forms of the signature peptidesare reported in Table 1. Data processing was performed with MassLynx 4.1 software. Each MS data point given in calibration curvesand sample analysis represents triplicate analyses by LC−MS/MS(MRM). Points are a measure of the peak area ratio of selecteddaughter ions of both the unlabeled and labeled peptide.

Article

data to the MASCOT search engine. Due to the lack of DNAinformation in the GeneBank, a phylogenetic tree was developedbased on a known protein sequence derived from cDNA, andthen the available species in the databases were used (Figure S1,Supporting Information). The protein identity of each band wasreported from the equivalent sequence of the closest species inthe database.

Tropomyosin Purification and Sequencing

■RESULTS

Protein Identification and Allergenicity Evaluation

Initial experiments were performed on shrimp meat, which wasisolated 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 afterovernight stirring in a suitable buffer at 4 °C, and then theproteins were profiled by SDS-PAGE. The allergenicity of eachprotein was examined using nine different sensitized patients’sera. Immunoblotting of the crude extract (Figure 2) shows the

The major allergen in shrimp, TM, was specifically targeted forpurification using multiple precipitation steps and then introducedto a recently developed protocol for primary structure determi-nation.19,20 This protocol is based on using multiple enzymaticdigestions, different MS ion sources, and a derivatization reactionto sequence the global protein with 100% amino acid coverage asshown in Figure 3. Three enzymes were used, trypsin, Gul-C V8,and ASP-N, to increase the diversity of the produced peptidesusing two MS ion sources, ESI and MALDI. The full amino acidsequence of TM was submitted to the UniProtKB/Swiss-Protdatabase with accession number P86704.1.

Arginine Kinase and Sarcoplasmic Ca-Binding ProteinPurification and Sequencing

Arginine kinase and SCBP were reported in several species asallergens,13,16,22,23,28 including northern shrimp. These proteinswere semipurified together because their isoelectric focusing valuesare fairly close to each other.23 In the present study, thepurification steps were monitored by SDS-PAGE as shown inFigure 4A, and the reactivity of the two proteins was examinedusing a pool of allergenic patient’s sera. The immunoblot of theSCBP shows a double band (Figure 4B) due to having severalisoforms that were confirmed later by MS. Ultimately, the aminoacid sequence coverage was 70% and 45% for SCBP and AK,respectively. Their amino acid sequence is reported in Figure 4Cand D, where the sequence information was combined fromseveral experimental approaches as described above.

Absolute Quantification Method Development

Figure 2. Clinical reactivity of nine shellfish allergic patients tonorthern shrimp (Pandalus borealis) crude extract using IgEimmunoblotting. The highlighted bands with boxes were labeledfrom 1 to 8, which are further analyzed by detailed proteomics.

various reactivity of each protein band with different patients.For instance, the 35 kDa band, a major allergenic protein,showed reactivity reached 100% (9/9) with different affinityresponses among patients. Expectedly, normal control serawere used for immunoblotting and did not show any binding(data not shown). The reactivity of each band with the patients’ sera wasreported in Table 2 as well as its identity which was achieved bypeptide mass fingerprinting (PMF). The equivalent band ofeach one that reacted was excised and tryptic digested and thenanalyzed by MALDI QToF mass spectrometry. The massspectral interpretation of each band was performed by uploading

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For quantitative analysis of targeted airborne allergens, anisotopic dilution tandem MS method was developed for TMand AK, where their signature peptides were assigned fromtheir protein sequence data. The criteria for selecting signaturepeptides were discussed elsewhere.29,30 Accordingly, the bestpeptide with the highest score of identity was reported for TMand AK in northern shrimp as SEEEVFGLQK andQQLVDDHFLFVSGDR, respectively. The signature peptideswere chemically synthesized, in both light and heavy forms, fordeveloping the proper MRM transitions of the triple quadru-pole mass spectrometer (Table 1). Representative product ionspectra for both peptides are shown in Figure 5, where themajor peptide fragment ions are shown for amino acidsequencing and confirming the identities of each peptide. Aqueous solutions of the signature peptides were used tooptimize the LC−MS parameters which enhance the product ionsignals for better sensitivity. Accordingly, the signature peptidemixture was chromatographed by a gradient reversed-phasemode to reach a limit of detection as low as 0.25 nM with linearcalibration curves ranging from 1 to 1000 nM (Figure 6B). Thereliability of the targeted quantification method was examined byusing shrimp crude extract solutions. These samples were trypticdigested as described in the method section and analyzed in thismethod. Representative chromatograms for a real extractedsample are shown in Figure 6A, where each transition represents


Table 2. List of the Reported North Shrimp Allergens That Have Been Identified Using Proteomics Mass Spectrometry in Crude Extract after Having Them Evaluated againstPatients' Sera

peptide sequencing

allergen code

100 22

31

214

49

10

gi|220172365gi|207298829gi|3907622

33

1004

100

22

gi|125995161

Figure 3

a

bandnumber

reactivity %

accession #

regular

protein name

size(kDa)

MASCOT score

sequencecoverage %

number of peptides

isoforms

5 and 1

Tropomyosin


4

Actin

Pandalus borealis northern shrimp (Pan b1)ref 22

5

Glyceraldehyde-3-phosphate dehydrogenase

refs 25, 26

44

37

123

32

9

gi|68272073gi|229256

651

8

Myosin heavy chain

Litopenaeus vannamei (pacific white shrimp); ref 27

11

227

98

6.7

11

gi|242006231

5

33 33

22 23

Arginine kinase

44

40

62 544 76

45 7027.8

14 9 5

gi|226693231

gi|238477327gi|136223

VAPEEHPVLLWEAPLNPK SYELPDGQVITISNERDITNYLGK SYELPDGQVITIGNERGYSFTTTAER EITGLAPSSIKEEYDESGPGIVHRK EITALAPSSIKSYELPDGQVITISNERAVFPSIVGREGYSFTTTAEREEYDESGPGIVHRGIDGFGR N/AHVYNEMKPENIPWSKGAGQNIIPSSTGAAKAGAHMKGGAKAGAEYIVESTGVFTTIEKAGAHMKGGAKLTGMAFRVPTPDVSVVDLTVRAGIQLSKLTQEAVADLER N/AELQARIEELLDEAGGATSAQIELNKDEAGGATSAQIELNKKRDLKLTQEAVDLLRQLEEAELQARIEELELSQVRQEILTQEAVADLERQIEEAEEIAALNLAKLADELRAEQEHAQTQEK Figure 4 (D)

Figure 4 (C)

N/A

3 3

Sarcoplasmic calcium-binding proteinTroponin C

Crangon crangon (North Sea shrimp) (Cra c2)Litopenaeus vannamei (White shrimp) (Lit v4)Crangon crangon (North Sea shrimp) (Cra c6)

2

Myosin light chain

Litopenaeus vannamei (White shrimp) (Lit v3)

44

19

51

4

1

gi|152013721

DYEINELNIQVNDLRDKKKLFEGGWFLIEEDEEALKTELRDEEALKTELRGLDPEALTGKHPPKEGFQLMDR

N/A

Article


a

Reactivity = number of subjects that react with the band/total number of subjects.


Figure 3. Full amino acid sequence of northern shrimp TM usingcomprehensive mass spectrometry. The different underlined proteinregions are derived from different proteases and/or MS ionizationapproaches. The chosen signature peptide is indicated by the blue box.

a signal for a specific signature peptide or one of their internalstandards.Shrimp Workplace Sampling and AnalysisDuring the shrimp fishing 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 PTFEfilters attached to air pumps. The air sample collection for theallergen profile 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 filters 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 subfield 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.32−35

Various epidemiological studies among seafood processorsindicate that the prevalence of occupational asthma is between2% and 36%, while it is more commonly associated withshellfish processing.19 The challenge for bioaerosol exposureassessment is the lack of methodological advancements in theaccurate and sensitive quantification of biomarker exposure.33

In the present study a novel method was developed to detectand quantify the most potent allergenic proteins from northernshrimp in air samples from shrimp processing workplaces. Several allergenic proteins are known to be unstable underheat and protease conditions. AK is one of these allergenswhich 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-specific IgE in the

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■

Figure 4. (A) Northern shrimp arginine kinase (AK) and sarcoplasmicCa-binding (SCBP) proteins purification steps, where crude extract(CE), 70% supernatant (70% S), 70% pellet (70% P), 90% supernatant(90% S), and 90% pellet (90% P) after ammonium sulfateprecipitation. (B) An immunoblot of both proteins against a pool ofpatients’ sera IgE, where the double bands represent the isoforms. (C)The amino acid sequence of SCBP and the detected isoform peptides.(D) The amino acid sequence of AK and the detected isoformpeptides. Note: In (C) and (D), red: covered experimentally, black:covered by similarity, and blue: isoform motifs.

patient serum were quantified using the ImmunoCAP system(Thermo Scientific). Total IgE ranged from 56 to 3401 kU/L,and shrimp-specific IgE ranged from 0.5 to 6.65 kU/L.



Figure 5. Product ion mass spectra of northern shrimp: (A) tropomyosin's signature peptide [SEEEVFGLQK] and (B) arginine kinase’s signaturepeptide (AK) [QQLVDDHFLFVSGDR].

The shrimp crude extract was successfully profiled in SDS-PAGE, which was enough to study its allergenicity againstpatients’ sera. As shown in Figure 2, a couple of bands (5 and 7)show reactivity with all patients’ sera, and therefore the contents ofthese bands are major allergen(s). Tropomyosin is a majorallergen in different seafood species, and its α-helix primarystructure is also known to develop hydrophobic interactions toform dimers and under certain conditions higher oligomers.20

Theidentity of these two bands was identified using MS, revealing theyare both related to TM. Normal control sera were used forimmunoblotting and did not show any binding (data not shown) Significant bands were also targeted for protein identificationusing MS, and the results are summarized in Table 2. Thereactivity of each band was calculated as the percentage of allergicto nonallergic patients. Most allergens exist in several variants(isoallergens), which are recognized differently by patient IgE, asshown in Figure 4A, where the allergenicity of SCBP against thepool of patient sera shows double bands.27,36−38 Noticeably,protein isoforms were detected in some of these allergens, whichare either related to different gene contributions in expression ordue to alternative splicing of the same unique gene.35,39

Table 2highlights the sites of heterogeneity for each isoallergen andreports their peptide sequences.

653

Tropomyosin and AK were both detected by MS in band 5.These proteins were purified, and their allergenicity wasconfirmed in separated forms. Tropomyosin is precipitatedout at 70% saturation by ammonium sulfate, while on the otherhand the AK precipitated at 90% saturation as seen in Figure4A. A complete amino acid sequence of TM was covered byMSMS using several proteases and ion sources. The TMstructure makes it very susceptible to the proteases and veryefficient in ionization.40 In contrast, AK and SCBP, globularproteins, show resistance to both Glu-C V8 and ASP-Ndigestion even after using RapiGest. The digestion was verypoor in the case of AK but relatively efficient in the case of TMand SCBP, which reflects the resulting sequence coverage. Tropomyosin has a highly conserved primary structure andshares a high amino acid sequence identity among crustaceans.The amino acid sequence identity of tropomyosin among eightdifferent shrimp species ranges from 96% to 100%. In this studyonly the major allergens, TM and AK, were targeted forquantification. The signature peptides for both allergens wereselected and evaluated based on selection criteria of signaturepeptides and examined by an NCBI protein blast testalgorithm.29,30 The method was developed to maintain the lowestlimit of detection to increase the sensitivity of routine analysis for



Figure 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 beable to draw a trend and propose preventative actions to avoidoccupational illness. The level of AK everywhere inside the plantwas slightly higher than TM, although the natural abundance ofTM in shrimp tissue is higher. This difference between AK andTM was also observed and discussed in previous studies in crabplants, where most of the AK comes from the hemolymph whichis easily aerosolized or steamed in cleaning and cooking areas.29,30

A bigger study is being conducted in Quebec-Canada followingthis approach, which we believe will allow us to propose curativeactions and help us to rationalize allergen exposure in differentworkstations.

Figure 7. Concentration of tropomyosin and arginine kinase in airsamples from three different workstations in a northern shrimpprocessing plant in the Province of Quebec. Number of samples in thepeeling station n = 4 and in the cooking and packing station n = 1each.

■ASSOCIATED CONTENTS

* Supporting Informationscreening workplaces. The method was very selective, reprodu-cible, and accurate for measuring the level of these allergens in airsamples (Figure 6). In terms of processing shrimp, the cooking and peeling stepsinvolve removing the shell from the cooked shrimp. The packingstep readies the cooked shrimp for consumption in an appropriatepackage for shipment. These processing steps were judged asbeing 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),and allergens (0.001−75.748 μg/m3).33 This study involved adeeper exploration and more specifics by reporting the mean levelsof indoor aerosolized allergens TM and AK: 125 and 480 ng/m3,respectively. On the basis of these results, it could be challengingto relocate a sensitized worker somewhere else in the plant to

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Figure S1. Phylogenetic tree based on the tropomyosin amino acidsequences for various crustacean species (accession number) andcompared to human and chicken tropomyosin American lobster(O44119); northern shrimp (P86704); Kuruma prawn(AB270630.1); Black tiger prawn (HM486525.1); Snow crab (A2V735); Horsehair crab (BAF47269); Cockroach (AAD19606);Locust (P31816); Dust mite (AAB69424); Storage mite (AAQ-54614); Pacific oyster (AAK96889); Blue mussel (AAA82259);Human (AAB59509); Chicken (AAA49112). This material isavailable free of charge via the Internet at http://pubs.acs.org.

■AUTHOR INFORMATION

Corresponding Author

*Phone: (+1) 416-586-4800. Ext 8268. Fax: (+1) 416-586-4200. E-mail: [email protected].

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTSThis research was partially funded by the National Sciences andEngineering Research Council (NSERC) and by the AustralianResearch Council (ARC)- Future Fellowship Award (Dr. AndreasLopata). We would like to acknowledge Memorial University ofNewfoundland (MUN) and the Department of Chemistry andIRSST for financial support. The clinical biochemistry lab inEastern Health (Dr. Edward Randell) and MUN Genomic andProteomics facility are highly acknowledged for giving access tothe mass spectrometry machines. Finally, the authors acknowl-edged Prof. Robyn O’Hehir (The Alfred Hospital, Melbourne,VIC, Australia) for supplying patient sera.

■Article

■REFERENCES

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656 dx.doi.org/10.1021/pr300755p | J. Proteome Res. 2013, 12, 647−656

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