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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
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

■ INTRODUCTION
Food allergy is one of the most common causes of anaphylaxis,
Double-blind placebo controlled food challenges (DBPCFCs)
were performed to determine threshold shrimp doses for
which is responsible for hundreds of fatalities annually worldwide. 1 individuals with shrimp allergies. The threshold doses ranged
Shrimp allergy axects about 2% of the general population world- from 14 to 16 g of shrimp equivalent to 32 mg of protein and
wide.2−4 In recent decades, seafood consumption has dramatically from 5 to 600 mg of protein for dixerent xsh. 4−7 Seafood allergen
increased as a healthier diet choice, which in turn has increased the quantixcation was initiated in 1997 by Lehrer’s group using a
number of people engaged in the seafood industry. These workers sandwich Enzyme-linked immunosorbent assay (ELISA) approach
are frequently exposed to aeroallergens that cause type-I hyper- for targeting brown shrimp tropomyosin (TM). 8 The detection
sensitivity. Northern shrimp (Pandalus borealis), caught in the limit was 4 ng/mL, and the assay was applied to detect TM in
North Atlantic and North Pacixc oceans, are the most commonly dixerent crustacean species such as crab and lobster. Recently, the
consumed shrimp in North America. The lack of molecular data method was optimized to evaluate the level of crustacean major
on allergenic proteins from most seafood species requires the
development of comprehensive strategies to study the biochemical
characteristics 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

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 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), chemiluminescent
ingestion-related allergic reactions,12 other allergens were substrate, sodium dodecylsulfate (SDS), ammonium formate, and
identixed and characterized such as arginine kinase (AK), 13,14 α-cyano-4-hydroxycinamic acid (HCCA) matrix were purchased
sarcoplasmic calcium-binding protein (SCBP), 15,16 and myosin- from Sigma-Aldrich (St. Louis, MO, USA). The Bradford assay kit
light 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 C18
ern shrimp (NS) as summarized in Figure 1.19 Sera from sensitized xlters were purchased for desalting from Millipore Corporation
patients were used to evaluate the proteomics proxle of NS which (Bedford, MA, USA). Powdered skimmed milk was purchased
was subsequently identixed using mass spectrometry. The identities from a local supermarket. Tris buxered saline (TBS) and phosphate
of 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 were
develop an absolute quantixcation (AQUA) MS approach. purchased from GeneMed Synthesis (San Francisco, CA, USA) as
The method reliability was estimated on real samples that were detailed in Table 1.
collected from a shrimp processing workplace, where allergen
levels 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 shrimp
Chemicals 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 left
All chemicals were used without further purixcation. Ammonium stirring overnight at 4 °C. The slurry was then centrifuged at
sulfate, 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 the
enzymes 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


Table 1. Standard Material Specixcations 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 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 for
developed 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 with
Sera from patients with shrimp allergies were used to demonstrate the same hybrid QqToF-MS/MS system equipped with a
the allergenicity of both the shrimp crude extract and purixed nanoelectrospray source (Protana XYZ manipulator). The
allergens. Patients were selected for this study based on clinical nanoelectrospray was generated from a PicoTip needle (10 μm
reactivity 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, and
Monash University as part of an ongoing survey. IgE antibody the resulting spectra were searched against the National Center
immunoblotting 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 (precursor
PVDF membrane.20 After blocking, the membranes were incubated and product ion mass tolerance set at 0.2 Da). Methionine
with patient serum (diluted 1:10 in 1% skimmed milk in PBS-T) oxidation was allowed as a variable modixcation and guanidinyl
overnight at 4 °C. The membrane was subsequently exposed to (K) as a xxed modixcation when the guanidation derivatization
rabbit polyclonal antihuman IgE antibody (DAKO, USA) and was performed. Peptides were considered identixed if the
goat antirabbit polyclonal antibody labeled with HRP (Promega, Mascot score was over a 95% conxdence limit.
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
Enzymatic Digestion the xshing season of 2011. The air samplers, Leland Legacy
The IgE antibody reactive protein bands were excised Sample Pump (SKC, Rochester, MN, USA), were deployed in
destained, and the protein trypsin was digested using a standard the peeling, cooking, and packing stations in addition to several
protocol.20 The tryptic peptides were extracted from the gel xeld blank samples collected outside the plants. The samplers
and 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 samples
enzymatic digestions, to increase the sequence coverage; trypsin, were collected on PTFE xlters at xow rates ranging from 2 to
Glu-C V8, or ASP-N enzymes were used in the presence of 3 L min−1. The xow rate of each sampler was calibrated before
RapiGest surfactant. Trypsin and Glu-C V8 enzymes were incubated and after collection using a Defender 510 air sampling pump
in 50 mM ABC overnight at 37 °C; however, the ASP-N enzymes calibrator (Air-Met Scientixc, Victoria, Australia). The xlters
were incubated in a reaction buxer: 50 mM Tris-HCl and 2.5 mM were subsequently shipped on dry ice to the lab and stored at
ZnSO4, 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 using
degrade 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 in
Mass Spectrometry Analysis for Allergen Characterization 100 μL of water and analyzed by LC−MS/MS.
The enzymatic peptides were analyzed using two dixerent ion
sources, MALDI and ESI, to increase the sequence coverage.
The MALDI targets were prepared following the double-layer Allergen Quantixcation by Mass Spectrometry
procedure detailed in another work.20The sample plate was The signature peptides of the major shrimp allergens were
analyzed in a MALDI-MS/MS at low-energy collision (CID)- determined (as described below) and chemically synthesized to
QSTAR XL hybrid quadrupole−quadrupole (Qq)/ToF-MS/ develop the following quantixcation method. Separation and
MS equipped with an o-MALDI ion source (Applied Biosystems, analysis were conducted using a Waters Alliance 2795 HPLC system
Foster 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 target
250 fmol enzymatic peptide sample was loaded onto a peptides. The peptides were separated on a reversed-phase
precolumn (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 gradient
Peptides 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 the
Packing, 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,



then 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 DNA
0.5 min (total: 11 min run time). A 20 μL injection was used at information in the GeneBank, a phylogenetic tree was developed
normal draw speed with a programmed washing procedure. The based on a known protein sequence derived from cDNA, and
eluted 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 was
through the capillary and orixce cone at 3.02 kV and 40 V, reported from the equivalent sequence of the closest species in
respectively. The precursor ions were fragmented using low-energy the database.
CID with argon gas and collision energy of 13 eV. The precursor
ions of the unlabeled and labeled forms of the signature peptides Tropomyosin Purixcation and Sequencing
are reported in Table 1. Data processing was performed with Mass
The major allergen in shrimp, TM, was specixcally targeted for
Lynx 4.1 software. Each MS data point given in calibration curves
purixcation using multiple precipitation steps and then introduced
and 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 enzymatic
daughter 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 peptides
Protein 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-Prot
Initial experiments were performed on shrimp meat, which was database with accession number P86704.1.
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 Arginine Kinase and Sarcoplasmic Ca-Binding Protein
overnight stirring in a suitable buxer at 4 °C, and then the Purixcation and Sequencing
proteins were proxled by SDS-PAGE. The allergenicity of each Arginine kinase and SCBP were reported in several species as
protein was examined using nine dixerent sensitized patients’ allergens,13,16,22,23,28 including northern shrimp. These proteins
sera. 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, for
Figure 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 ion
immunoblotting. The highlighted bands with boxes were labeled spectra for both peptides are shown in Figure 5, where the
from 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 to
various reactivity of each protein band with dixerent patients.
optimize the LC−MS parameters which enhance the product ion
For instance, the 35 kDa band, a major allergenic protein,
signals for better sensitivity. Accordingly, the signature peptide
showed reactivity reached 100% (9/9) with dixerent axnity
mixture was chromatographed by a gradient reversed-phase
responses among patients. Expectedly, normal control sera
mode to reach a limit of detection as low as 0.25 nM with linear
were 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 tryptic
reported in Table 2 as well as its identity which was achieved by
digested as described in the method section and analyzed in this
peptide mass xngerprinting (PMF). The equivalent band of
method. Representative chromatograms for a real extracted
each one that reacted was excised and tryptic digested and then
sample are shown in Figure 6A, where each transition represents
analyzed by MALDI QToF mass spectrometry. The mass
spectral interpretation of each band was performed by uploading



Table 2. List of VAPEEHPVLLWEAPLNPK
the Reported SYELPDGQVITISNER
North Shrimp DITNYLGK
isoforms SYELPDGQVITIGNER
Allergens That GYSFTTTAER
Have Been EITGLAPSSIK
Identixed EEYDESGPGIVHRK
Using peptide EITALAPSSIK N/A N/A
sequencing SYELPDGQVITISNERAVFPSIVGREGYSFTTTAEREEYDESGPGIVHRGIDGFGR Figure 4 (C)
Proteomics N/A
Figure 3
Mass HVYNEMKPENIPWSKGAGQNIIPSSTGAAKAGAHMKGGAKAGAEYIVESTGVFTTIEKAGAHMKGGAKLTGMAF
Spectrometry RVPTPDVSVVDLTVRAGIQLSKLTQEAVADLER
N/A
in Crude ELQARIEELLDEAGGATSAQIELNKDEAGGATSAQIELNKKRDLKLTQEAVDLLRQLEEAELQARIEELELSQVRQ DYEINELNIQVNDLRDKKKLF
Extract after EILTQEAVADLERQIEEAEEIAALNLAKLADELRAEQEHAQTQEK EGGWFLIEEDEEALKTELRDE
Having Them regular Figure 4 (D) EALKTELRGLDPEALTGKHPP
KEGFQLMDR
Evaluated
againstPatients'
Sera

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



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

a signal for a specixc signature peptide or one of their internal
standards.
Shrimp Workplace Sampling and Analysis
During the shrimp xshing 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
xlters attached to air pumps. The air sample collection for the
allergen proxle 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 xlters 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.

■ DISCUSSION
Allergenomics is a subxeld 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 under-
recognized, 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 Figure 4. (A) Northern shrimp arginine kinase (AK) and sarcoplasmic
shellxsh processing.19 The challenge for bioaerosol exposure Ca-binding (SCBP) proteins purixcation steps, where crude extract
assessment is the lack of methodological advancements in the (CE), 70% supernatant (70% S), 70% pellet (70% P), 90% supernatant
accurate and sensitive quantixcation of biomarker exposure. 33 (90% S), and 90% pellet (90% P) after ammonium sulfate
In the present study a novel method was developed to detect precipitation. (B) An immunoblot of both proteins against a pool of
and 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 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-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.



Figure 5. Product ion mass spectra of northern shrimp: (A) tropomyosin's signature peptide [SEEEVFGLQK] and (B) arginine kinase’s signature
peptide (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 was
patients’ sera. As shown in Figure 2, a couple of bands (5 and 7) conxrmed in separated forms. Tropomyosin is precipitated
show reactivity with all patients’ sera, and therefore the contents of out at 70% saturation by ammonium sulfate, while on the other
these bands are major allergen(s). Tropomyosin is a major hand the AK precipitated at 90% saturation as seen in Figure
allergen in dixerent seafood species, and its α-helix primary 4A. A complete amino acid sequence of TM was covered by
structure is also known to develop hydrophobic interactions to MSMS using several proteases and ion sources. The TM
form dimers and under certain conditions higher oligomers. 20 The structure makes it very susceptible to the proteases and very
identity of these two bands was identixed using MS, revealing they excient in ionization.40 In contrast, AK and SCBP, globular
are both related to TM. Normal control sera were used for proteins, show resistance to both Glu-C V8 and ASP-N
immunoblotting 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 TM
using 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 and
to 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 eight
shown in Figure 4A, where the allergenicity of SCBP against the dixerent shrimp species ranges from 96% to 100%. In this study
pool of patient sera shows double bands.27,36−38 Noticeably, only the major allergens, TM and AK, were targeted for
protein isoforms were detected in some of these allergens, which quantixcation. The signature peptides for both allergens were
are either related to dixerent gene contributions in expression or selected and evaluated based on selection criteria of signature
due to alternative splicing of the same unique gene. 35,39 Table 2 peptides and examined by an NCBI protein blast test
highlights the sites of heterogeneity for each isoallergen and algorithm.29,30 The method was developed to maintain the lowest
reports their peptide sequences. limit 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 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 air
samples from three dixerent workstations in a northern shrimp
processing plant in the Province of Quebec. Number of samples in the
■
S
ASSOCIATED CONTENT
peeling station n = 4 and in the cooking and packing station n = 1
each. * 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
screening workplaces. The method was very selective, reprodu-
(O44119); northern shrimp (P86704); Kuruma prawn
cible, and accurate for measuring the level of these allergens in air
(AB270630.1); Black tiger prawn (HM486525.1); Snow crab (A2
samples (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 is
package 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 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),
■ AUTHOR INFORMATION
Corresponding Author
and allergens (0.001−75.748 μg/m 3).33 This study involved a
deeper 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 challenging
to relocate a sensitized worker somewhere else in the plant to Notes
The authors declare no competing xnancial interest.



■ ACKNOWLEDGMENTS
This 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 novel
Research 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 by
Proteomics facility are highly acknowledged for giving access to adults suggests that shrimp sensitization decreases with age. J. Allergy
the 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-
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