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Comprehensive Proteomics Approach in Characterizing and Quantifying Allergenic Proteins from Northern Shrimp: Toward Better Occupational Asthma Prevention Anas 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 Cook University, 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 different seafood. This work involves the application of an allergenomics strategy to study the most potent allergens of northern shrimp. The proteins were extracted from shrimp tissue and profiled by gel electrophoresis. Allergenic proteins were identified based on their reactivity to patient sera and were structurally identified using tandem mass spectrometry. Northern shrimp tropomyosin, arginine kinase, and sarcoplasmic calcium-binding protein were found to be the most significant 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 quantified using tandem mass specrometry. Allergens were detected in all areas of the plant, reaching levels as high as 375 and 480 ng/m3 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

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INTRODUCTION Food allergy is one of the most common causes of anaphylaxis, which is responsible for hundreds of fatalities annually worldwide.1 Shrimp allergy affects about 2% of the general population worldwide.2−4 In recent decades, seafood consumption has dramatically increased as a healthier diet choice, which in turn has increased the number of people engaged in the seafood industry. These workers are frequently exposed to aeroallergens that cause type-I hypersensitivity. Northern shrimp (Pandalus borealis), caught in the North Atlantic and North Pacific oceans, are the most commonly consumed shrimp in North America. The lack of molecular data on allergenic proteins from most seafood species requires the development of comprehensive strategies to study the biochemical characteristics of these allergens. © 2012 American Chemical Society

Double-blind placebo controlled food challenges (DBPCFCs) were performed to determine threshold shrimp doses for individuals with shrimp allergies. The threshold doses ranged from 14 to 16 g of shrimp equivalent to 32 mg of protein and from 5 to 600 mg of protein for different fish.4−7 Seafood allergen quantification was initiated in 1997 by Lehrer’s group using a sandwich Enzyme-linked immunosorbent assay (ELISA) approach for targeting brown shrimp tropomyosin (TM).8 The detection limit was 4 ng/mL, and the assay was applied to detect TM in different crustacean species such as crab and lobster. Recently, the method was optimized to evaluate the level of crustacean major Received: August 8, 2012 Published: 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 molecular characterization.17

allergen, TM, in processed food9,10 with a limit of detection of about 2.5 mg/kg.11 Although TM is the major crustacean allergen responsible for ingestion-related allergic reactions,12 other allergens were identified and characterized such as arginine kinase (AK),13,14 sarcoplasmic calcium-binding protein (SCBP),15,16 and myosinlight chain (MLC).17,18 This study introduces a functional proteomics strategy (allergenomic) to evaluate the potential allergenic proteins in northern shrimp (NS) as summarized in Figure 1.19 Sera from sensitized patients were used to evaluate the proteomics profile of NS which was subsequently identified using mass spectrometry. The identities of detected allergens were confirmed by comparing the MS results with other species available in GenBank. Selected allergens were then purified and sequenced to prove the validity of the strategy for allergen identification. TM, AK, and SCBP were determined to be the most significant allergens and thus were purified and sequenced. The signature peptides for each allergen were assigned and determined to develop an absolute quantification (AQUA) MS approach. The method reliability was estimated on real samples that were collected from a shrimp processing workplace, where allergen levels were noticeably high in the main processing station.

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ethylenediaminetetraacetic acid (EDTA), formic acid (FA), ammonium bicarbonate, o-ethylisourea hemisulfate, ammoniumhydroxide, horseradish peroxidase (HRP), chemiluminescent substrate, sodium dodecylsulfate (SDS), ammonium formate, and α-cyano-4-hydroxycinamic acid (HCCA) matrix were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Bradford assay kit and PVDF immunoblot membranes were purchased from BioRad (Hercules, CA, USA), and dialysis bags were purchased from Fischer Scientific (Roncho Dominguez, CA, USA). ZipTip C18 filters were purchased for desalting from Millipore Corporation (Bedford, MA, USA). Powdered skimmed milk was purchased from a local supermarket. Tris buffered saline (TBS) and phosphate buffered 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 were purchased from GeneMed Synthesis (San Francisco, CA, USA) as detailed in Table 1. Northern Shrimp Extracts

Fresh northern shrimp were collected from a fishing boat in St. John’s−NL Canada. After shell removal, the meat was rinsed with water and stored in liquid nitrogen. Five grams of shrimp was homogenized with 50 mL of buffer A (1 M KCl, 25 mM Tris-HCl, pH 8.0, 0.25 M DTT, and 0.5 mM EDTA) and left stirring overnight at 4 °C. The slurry was then centrifuged at 10 000 rpm for 30 min at 4 °C. The total protein concentration was determined using the Bradford assay. The crude extract was used for further characteizing

MATERIALS AND METHODS

Chemicals and Materials

All chemicals were used without further purification. Ammonium sulfate, acetonitrile (ACN), hydrochloric acid, and methanol were supplied by ACP (Montreal, Canada). Trypsin sequencing grade enzymes were purchased from Promega (WI, USA). Tris(hydroxymethyl) aminomethane (Tris), dithiotheritol (DTT), 648



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

peptide code

purity %

average mass

SEEEVFGLQK SEEE(d8-V)FGLQK QQLVDDHFLFVSGDR QQL(d8-V)DDHFLF(d8-V)SGDR

TM d8-TM AK d16-AK

98.25 98.25 98.27 98.34

1165.8 1173.7 1776.1 1791.1

Q1(z) m/z 583 587 592 598

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

Q3(ion) m/z 217 217 586 592

(b9) (b9) ([M−H2O+3H]+3) ([M−H2O+3H]+3)

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 for 55 min, 60−90% for 3 min, and 90% B for 5 min was applied. Including a regeneration step, one run was 106 min long. The ESI−MS spectra of the LC-eluting peptides were measured with the same hybrid QqToF-MS/MS system equipped with a nanoelectrospray source (Protana XYZ manipulator). The nanoelectrospray was generated from a PicoTip needle (10 μm i.d., New Objectives, Wobum, MA, USA) at a voltage of 2400 V. The samples were further analyzed by CID−MS/MS, and the resulting spectra were searched against the National Center for Biotechnology Information nonredundant (NCBInr) database using a Matrix Science (Mascot) search engine (precursor and product ion mass tolerance set at 0.2 Da). Methionine oxidation was allowed as a variable modification and guanidinyl (K) as a fixed modification when the guanidation derivatization was performed. Peptides were considered identified if the Mascot score was over a 95% confidence limit.

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

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

Air Sample Collection

Air samples were collected from a northern shrimp plant during the fishing season of 2011. The air samplers, Leland Legacy Sample Pump (SKC, Rochester, MN, USA), were deployed in the peeling, cooking, and packing stations in addition to several field blank samples collected outside the plants. The samplers were programmed to collect particulates for an 8-h working shift, where the personal breathing zone (PBZ) air samples were collected on PTFE filters at flow rates ranging from 2 to 3 L min−1. The flow rate of each sampler was calibrated before and after collection using a Defender 510 air sampling pump calibrator (Air-Met Scientific, Victoria, Australia). The filters were subsequently shipped on dry ice to the lab and stored at −80 °C until protein extraction. The proteins were extracted from the PTFE filters using 0.05% 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 digestion as described above. Finally, the peptides were reconstituted in 100 μL of water and analyzed by LC−MS/MS.

Enzymatic Digestion

The IgE antibody reactive protein bands were excised destained, and the protein trypsin was digested using a standard protocol.20 The tryptic peptides were extracted from the gel and desalted using C18 ZipTip for MALDI-QqToF analyses. The purified proteins were exposed to several in-solution enzymatic digestions, to increase the sequence coverage; trypsin, Glu-C V8, or ASP-N enzymes were used in the presence of RapiGest surfactant. Trypsin and Glu-C V8 enzymes were incubated in 50 mM ABC overnight at 37 °C; however, the ASP-N enzymes were incubated in a reaction buffer: 50 mM Tris-HCl and 2.5 mM ZnSO4, at pH 8 overnight at 37 °C. The in-solution digestion samples were quenched using the equivalent volume of 1% TFA to degrade the acid labile surfactant, and then the samples were freezedried and stored at −80 °C before MS analyses. Mass Spectrometry Analysis for Allergen Characterization

The enzymatic peptides were analyzed using two different ion sources, MALDI and ESI, to increase the sequence coverage. The MALDI targets were prepared following the double-layer procedure detailed in another work.20The sample plate was analyzed 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 DIONEX UltiMate3000 Nano LC System (Germering, Germany). A 250 fmol enzymatic peptide sample was loaded onto a precolumn (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 (LC Packing, Sunnyvale, CA)) at 180 nL/min using the following gradient. The aqueous mobile phases consisted of (A) 0.1%

Allergen Quantification by Mass Spectrometry

The signature peptides of the major shrimp allergens were determined (as described below) and chemically synthesized to develop the following quantification method. Separation and analysis were conducted using a Waters Alliance 2795 HPLC system coupled to a Micromass Quattro Ultima (Water Corporation, Milford, MA, USA) LC-MSMS operated in electrospray positiveionization (ESI) mode and adjusted to separate the target peptides. The peptides were separated on a reversed-phase chromatography column (Kinetex C18, 2.1 mm × 100 mm, 2.6 μM particle size, Phenomenex, CA, USA) at 20 °C. A gradient elution was performed, where the aqueous mobile phase (A) consisted of HPLC-grade water with 0.1% formic acid and the organic 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, 649



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data to the MASCOT search engine. Due to the lack of DNA information in the GeneBank, a phylogenetic tree was developed based on a known protein sequence derived from cDNA, and then the available species in the databases were used (Figure S1, Supporting Information). The protein identity of each band was reported from the equivalent sequence of the closest species in the database.

then 90% B for 3 min after which it reverted back to the 5% for 0.5 min (total: 11 min run time). A 20 μL injection was used at normal draw speed with a programmed washing procedure. The eluted peptides were desolvated during ESI with a gas flow rate of 400 L h−1 and a temperature of 250 °C. The ions were accelerated through the capillary and orifice cone at 3.02 kV and 40 V, respectively. The precursor ions were fragmented using low-energy CID with argon gas and collision energy of 13 eV. The precursor ions of the unlabeled and labeled forms of the signature peptides are reported in Table 1. Data processing was performed with Mass Lynx 4.1 software. Each MS data point given in calibration curves and sample analysis represents triplicate analyses by LC−MS/MS (MRM). Points are a measure of the peak area ratio of selected daughter ions of both the unlabeled and labeled peptide.

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Tropomyosin Purification and Sequencing

The major allergen in shrimp, TM, was specifically targeted for purification using multiple precipitation steps and then introduced to a recently developed protocol for primary structure determination.19,20 This protocol is based on using multiple enzymatic digestions, different 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, and ASP-N, to increase the diversity of the produced peptides using two MS ion sources, ESI and MALDI. The full amino acid sequence of TM was submitted to the UniProtKB/Swiss-Prot database with accession number P86704.1.

RESULTS

Protein Identification and Allergenicity Evaluation

Initial experiments were performed on shrimp meat, which was isolated from freshly caught shrimp previously snap frozen in liquid nitrogen to quench any protease activities and then stored at −80 °C. A shrimp crude extract was collected after overnight stirring in a suitable buffer at 4 °C, and then the proteins were profiled by SDS-PAGE. The allergenicity of each protein was examined using nine different sensitized patients’ sera. Immunoblotting of the crude extract (Figure 2) shows the

Arginine Kinase and Sarcoplasmic Ca-Binding Protein Purification and Sequencing

Arginine kinase and SCBP were reported in several species as allergens,13,16,22,23,28 including northern shrimp. These proteins were semipurified together because their isoelectric focusing values are fairly close to each other.23 In the present study, the purification 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 confirmed 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 Quantification 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, for developing the proper MRM transitions of the triple quadrupole mass spectrometer (Table 1). Representative product ion spectra for both peptides are shown in Figure 5, where the major peptide fragment ions are shown for amino acid sequencing and confirming the identities of each peptide. Aqueous solutions of the signature peptides were used to optimize the LC−MS parameters which enhance the product ion signals for better sensitivity. Accordingly, the signature peptide mixture was chromatographed by a gradient reversed-phase mode to reach a limit of detection as low as 0.25 nM with linear calibration curves ranging from 1 to 1000 nM (Figure 6B). The reliability of the targeted quantification method was examined by using shrimp crude extract solutions. These samples were tryptic digested as described in the method section and analyzed in this method. Representative chromatograms for a real extracted sample are shown in Figure 6A, where each transition represents

Figure 2. Clinical reactivity of nine shellfish allergic patients to northern shrimp (Pandalus borealis) crude extract using IgE immunoblotting. The highlighted bands with boxes were labeled from 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 affinity responses among patients. Expectedly, normal control sera were used for immunoblotting and did not show any binding (data not shown). The reactivity of each band with the patients’ sera was reported in Table 2 as well as its identity which was achieved by peptide mass fingerprinting (PMF). The equivalent band of each one that reacted was excised and tryptic digested and then analyzed by MALDI QToF mass spectrometry. The mass spectral interpretation of each band was performed by uploading 650


Glyceraldehyde-3-phosphate dehydrogenase

5

651

Myosin light chain

3

2

Litopenaeus vannamei (White shrimp) (Lit v3)

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

Litopenaeus vannamei (pacific white shrimp); ref 27

refs 25, 26

100

Pandalus borealis northern shrimp (Pan b1) ref 22

44

33

33

44

11

44

22

reactivity %

a

allergen code

19

23

22

40

227

37

31

33

size (kDa)

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

Sarcoplasmic calcium-binding protein Troponin C

3

a

Arginine kinase

5

Myosin heavy chain

Actin

4

8

Tropomyosin

protein name

5 and 1

band number

51

76

544

62

98

123

214

1004

MASCOT score

4

27.8

70

45

6.7

32

49

100

sequence coverage %

1

5

9

14

11

9

10

22

number of peptides accession #

gi|152013721

gi|136223

gi|238477327

gi|226693231

gi|242006231

gi|68272073 gi|229256

gi|220172365 gi|207298829 gi|3907622

gi|125995161

Figure 3

isoforms

DYEINELNIQVNDLR DKKKLFEGGW FLIEEDEEALKTELR DEEALKTELR GLDPEALTGKHPPK EGFQLMDR

N/A

N/A

Figure 4 (C)

VAPEEHPVLLWEAPLNPK SYELPDGQVITISNER DITNYLGK SYELPDGQVITIGNER GYSFTTTAER EITGLAPSSIK EEYDESGPGIVHRK EITALAPSSIK SYELPDGQVITISNER AVFPSIVGR EGYSFTTTAER EEYDESGPGIVHR GIDGFGR N/A HVYNEMKPENIPWSK GAGQNIIPSSTGAAK AGAHMKGGAK AGAEYIVESTGVFTTIEK AGAHMKGGAK LTGMAFR VPTPDVSVVDLTVR AGIQLSK LTQEAVADLER N/A ELQARIEEL LDEAGGATSAQIELNK DEAGGATSAQIELNKKR DLKLTQEAV DLLRQLEEA ELQARIEEL ELSQVRQEI LTQEAVADLER QIEEAEEIAALNLAK LADELRAEQEHAQTQEK Figure 4 (D)

regular

peptide sequencing

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

Journal of Proteome Research Article



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Figure 3. Full amino acid sequence of northern shrimp TM using comprehensive mass spectrometry. The different underlined protein regions are derived from different proteases and/or MS ionization approaches. The chosen signature peptide is indicated by the blue box.

a signal for a specific signature peptide or one of their internal standards. Shrimp Workplace Sampling and Analysis

During the shrimp fishing season in the summer of 2011, a processing plant located on the northern shore of the Province of Quebec, Canada, was targeted to be a model for our approach. Personal breathing zone air samples were collected using PTFE filters attached to air pumps. The air sample collection for the allergen profile was recently standardized and has been followed in this study.29,30 The samplers were deployed on workers toiling in cooking, peeling, and packing areas for 8 h of operation and shrimp processing. The target allergens were extracted from the filters using a standard protocol, and the eluted allergens were tryptic digested and then analyzed by LC−MS/MS.29,30 The levels of both targeted allergens (TM and AK) were reported in Figure 7.

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DISCUSSION Allergenomics is a subfield of proteomics where the reactive proteins, along with human sera’s IgE, are screened and targeted for further characterization (Figure 1). Recent studies on the global burden of disease indicate that occupational lung diseases are caused by exposure to airborne agents such as allergens. In addition, it is suggested that up to 15% of adult asthma is attributed to occupational exposure.31 While occupational respiratory diseases are still largely underrecognized, they remain poorly diagnosed and managed.32−35 Various epidemiological studies among seafood processors indicate that the prevalence of occupational asthma is between 2% and 36%, while it is more commonly associated with shellfish processing.19 The challenge for bioaerosol exposure assessment is the lack of methodological advancements in the accurate and sensitive quantification of biomarker exposure.33 In the present study a novel method was developed to detect and quantify the most potent allergenic proteins from northern shrimp in air samples from shrimp processing workplaces. Several allergenic proteins are known to be unstable under heat and protease conditions. AK is one of these allergens which entails working with fresh meat stored at −80 °C after snap freezing them in liquid nitrogen. For this study, the patients were recruited based on their clinical history of reactivity to shrimp. Total IgE and shrimp-specific IgE in the

Figure 4. (A) Northern shrimp arginine kinase (AK) and sarcoplasmic Ca-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 sulfate precipitation. (B) An immunoblot of both proteins against a pool of patients’ 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 isoform peptides. 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. 652



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

Tropomyosin and AK were both detected by MS in band 5. These proteins were purified, and their allergenicity was confirmed in separated forms. Tropomyosin is precipitated out at 70% saturation by ammonium sulfate, while on the other hand the AK precipitated at 90% saturation as seen in Figure 4A. A complete amino acid sequence of TM was covered by MSMS using several proteases and ion sources. The TM structure makes it very susceptible to the proteases and very efficient in ionization.40 In contrast, AK and SCBP, globular proteins, show resistance to both Glu-C V8 and ASP-N digestion even after using RapiGest. The digestion was very poor in the case of AK but relatively efficient in the case of TM and SCBP, which reflects the resulting sequence coverage. Tropomyosin has a highly conserved primary structure and shares a high amino acid sequence identity among crustaceans. The amino acid sequence identity of tropomyosin among eight different shrimp species ranges from 96% to 100%. In this study only the major allergens, TM and AK, were targeted for quantification. The signature peptides for both allergens were selected and evaluated based on selection criteria of signature peptides and examined by an NCBI protein blast test algorithm.29,30 The method was developed to maintain the lowest limit of detection to increase the sensitivity of routine analysis for

The shrimp crude extract was successfully profiled in SDSPAGE, which was enough to study its allergenicity against patients’ sera. As shown in Figure 2, a couple of bands (5 and 7) show reactivity with all patients’ sera, and therefore the contents of these bands are major allergen(s). Tropomyosin is a major allergen in different seafood species, and its α-helix primary structure is also known to develop hydrophobic interactions to form dimers and under certain conditions higher oligomers.20 The identity of these two bands was identified using MS, revealing they are both related to TM. Normal control sera were used for immunoblotting and did not show any binding (data not shown) Significant bands were also targeted for protein identification using MS, and the results are summarized in Table 2. The reactivity of each band was calculated as the percentage of allergic to nonallergic patients. Most allergens exist in several variants (isoallergens), which are recognized differently by patient IgE, as shown in Figure 4A, where the allergenicity of SCBP against the pool of patient sera shows double bands.27,36−38 Noticeably, protein isoforms were detected in some of these allergens, which are either related to different gene contributions in expression or due to alternative splicing of the same unique gene.35,39 Table 2 highlights the sites of heterogeneity for each isoallergen and reports their peptide sequences. 653



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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 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 difference 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 different workstations.

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

ASSOCIATED CONTENT

S 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 lobster (O44119); northern shrimp (P86704); Kuruma prawn (AB270630.1); Black tiger prawn (HM486525.1); Snow crab (A2 V735); Horsehair crab (BAF47269); Cockroach (AAD19606); Locust (P31816); Dust mite (AAB69424); Storage mite (AAQ54614); Pacific oyster (AAK96889); Blue mussel (AAA82259); Human (AAB59509); Chicken (AAA49112). This material is available free of charge via the Internet at http://pubs.acs.org.

screening workplaces. The method was very selective, reproducible, and accurate for measuring the level of these allergens in air samples (Figure 6). In terms of processing shrimp, the cooking and peeling steps involve removing the shell from the cooked shrimp. The packing step readies the cooked shrimp for consumption in an appropriate package for shipment. These processing steps were judged as being the most relevant to conduct air monitoring. The air samples were collected during 8 h of operation and processing. Jeebhay and Cartier (2010) surveyed several studies and found 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 a deeper exploration and more specifics by reporting the mean levels of indoor aerosolized allergens TM and AK: 125 and 480 ng/m3, respectively. On the basis of these results, it could be challenging to relocate a sensitized worker somewhere else in the plant to

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+1) 416-586-4800. Ext 8268. Fax: (+1) 416-5864200. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 654



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ACKNOWLEDGMENTS This research was partially funded by the National Sciences and Engineering Research Council (NSERC) and by the Australian Research Council (ARC)- Future Fellowship Award (Dr. Andreas Lopata). We would like to acknowledge Memorial University of Newfoundland (MUN) and the Department of Chemistry and IRSST for financial support. The clinical biochemistry lab in Eastern Health (Dr. Edward Randell) and MUN Genomic and Proteomics facility are highly acknowledged for giving access to the mass spectrometry machines. Finally, the authors acknowledged Prof. Robyn O’Hehir (The Alfred Hospital, Melbourne, VIC, Australia) for supplying patient sera.

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