Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospary ionization and matrix-assisted laser desorption ionization QqToF tandemmass spectrometry

Article abstract of DOI:10.1002/jms.1721

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) using hybrid quadruple time-of-flight tandem mass spectrometry (QqToF-MS). The native polypeptidemolecular weight of TM was determined to be 32 733 Da. The protein was further characterized using the 'bottomup' MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post-translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TMwas identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI-QqToF-MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place. Copyright

Full text of DOI:10.1002/jms.1721

Research Article
Received: 5 November 2009
Accepted: 26 January 2010
Published online in Wiley Interscience: 2 March 2010
(www.interscience.com) DOI 10.1002/jms.1721
Characterization and de novo sequencing of
snow crab tropomyosin enzymatic peptides by
both electrospary ionization and
matrix-assisted laser desorption ionization
QqToF tandem mass spectrometry
Anas M. Abdel Rahman,a∗ Andreas L. Lopata,b Robyn E. O’Hehir,c
John J. Robinson,d Joseph H. Banoubd,e and Robert J. Helleura
The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an
occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM
protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix-assisted
laser desorption/ionization (MALDI) using hybrid quadruple time-of-flight tandem mass spectrometry (QqToF-MS). The native
polypeptide molecular weight of TM was determined to be 32 733 Da. The protein was further characterized using the ‘bottom-
up’ MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing
of the most abundant peptides performed. Any post-translational modifications were identified by searching their calculated
and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated
using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide
for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The
identification of a signature peptide for the allergen TM using MALDI-QqToF-MS will be critical for the sensitive and specific
c
quantification of this highly allergenic protein in the work place. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Keywords: snow crab; tropomyosin; de novo sequencing; mass spectrometry; allergen; signature peptide; in-gel guanidation; PTM
evaluation
Introduction
of TM.[9] The TM muscle isoform contains 284 amino acids after
including a highly conserved N-terminal region.[10–12]
Seafood plays an important role in human nutrition and
health. The growing international trade in seafood reflects
the popularity and frequency of consumption of a variety of
seafood products across many countries. Unfortunately, increased
production and consumption of seafood has resulted in more
frequent adverse health problems (i.e. food allergies) among
consumers of seafood.[1] The fishing and fish processing industry
has experienced tremendous growth in recent years with over
41 million workers worldwide engaged in various activities and
exposed to seafood.[2]
Increased levels of production and processing of seafood
have led and continue to lead to more frequent reporting of
occupational health problems such as asthma and other allergic
reactions, particularly, in the crustacean processing sector.[3,4]
Tropomyosin (TM) is one of the common muscle proteins that
mediate the interaction between the troponin complex and actin,
which regulates muscle contraction.[5] Crustaceans’ TM was first
identifiedinshrimpbyHoffmanet al. in1981.[6] Itisawater-soluble
and heat-stable protein with molecular weights (MWs) ranging
between 34 and 39 kDa.[7] It has a highly conserved amino acid
sequence among different invertebrate organisms and is present
in muscle as well as non-muscle cells.[8] Differential splicing of the
pre-messenger ribonucleic acid (pre-mRNA) produces isoforms
Research has shown that TM isolated from shrimps and crabs
is a major food allergen.[13] Snow crab is among the sea foods
that are most frequently associated with an Immunoglobulin E
(IgE) mediated type I hypersensitivity in patients with food allergy.
Urticaria, asthma and diarrhea are the major clinical symptoms
that are caused by type I hypersensitivity.[14]
∗
Correspondence to: Anas M. Abdel Rahman, Department of Chemistry, Memo-
rial University of Newfoundland, St John’s, Newfoundland, Canada A1B 3X7.
E-mail: anasar@mun.ca
a
b
c
Department of Chemistry, Memorial University of Newfoundland, St John’s,
Newfoundland, Canada A1B 3X7
School of Applied Science, Marine Biomedical Sciences and Health Research
Group, RMIT University, Bundoora, 3083 Victoria, Australia
Department of Allergy, Immunology and Respiratory Medicine, Alfred Hospital
and Monash University, Melbourne, 3004 Victoria, Australia
d
e
Department of Biochemistry, Memorial University of Newfoundland, St John’s,
Newfoundland, Canada A1B 3X9
Fisheries and Oceans Canada, Science Branch, Special Projects, St John’s,
Newfoundland, Canada A1C 5X1
c
J. Mass. Spectrom. 2010, 45, 372–381
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides
Monitoring the level of TM in the snow crab processing
workplaces is essential in reducing the workers’ risk of devel-
oping allergic airway disease. The total amount of snow crab
allergens have previously been characterized and measured
through immunological reactivity by enzyme-linked immunosor-
bent assay (ELISA),[15] radioallergosorbent test (RAST),[13] and
immunoblotting.[15,16] The main disadvantage in analyzing the
total crab protein by conventional methods is the inclusion of
additional proteins that are not necessarily involved in the allergic
reactions of the exposed worker.[17] However, by targeting and
quantifying the major crustacean allergen protein, one can
correlate the amount of allergen with the severity of the allergen
and determine the threshold values.[3]
Inthepresentstudy, thesnowcrabTMwasisolatedandpurified.
For studying different isoforms of TM, tissues were obtained from
claw and leg and the extracted proteins were separated by sodium
dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) to
yield homogeneous bands. Purified TM was used in western blot
analysis to study the reactivity of the TM with specific reactivity of
monoclonal and polyclonal antibodies as well as serum of patients
with crustacean allergy.
(FA), ammonium bicarbonate, o-methylisourea hemisulfate, am-
monium hydroxide, horse radish peroxidase (HRP), 3,3ꢁ,5,5ꢁ-
tetramethylbenzidine (TMB) and α-cyano-4-hydroxycinamic acid
(HCCA) matrix were from Sigma-Aldrich (St Louis, MO, USA). The
Bradford assay kit was from BioRad (Hercules, CA, USA). Dialysis
bags were from Fischer Scientific (Roncho Dominguez, CA, USA).
For desalting, the ZipTip C18 filters were purchased from Millipore
Corporation (Bedford, MA, USA)
TM purification
Acetone powder extract
The tissue of the claw and legs (∼500 g) was removed from fresh
frozen crab sections, homogenized in a precooled stainless steel
homogenizer (Polytron, Brinkmann instruments) and then mixed
with an equal volume of deionized water for 2 min. After being
allowed to stand for 20 min, the mixture was strained through two
layers of cheesecloth. The crude protein was washed with 500 ml
95% ethanol for 3 min, washed again with 50% ethanol followed
by two times with 95% ethanol and finally washed two times with
acetone.
TheMWoftheextractedTMwasdeterminedbyelectrosprayion-
ization quadruple time-of-flight tandem mass spectrometry (ESI-
QqToF). Following fractionation by SDS-PAGE, gel bands were ex-
cised and subjected to enzymatic digestion. The peptides were an-
alyzed using peptide mass fingerprinting (PMF) by matrix-assisted
laserdesorption/ionization(MALDI)-QqToF-MS.Peptideswerealso
de novo sequenced using the ‘bottom-up’ MS approach.[17–20]
It has been well established that PMF and the various out-
comes of the MS approach can give complete information about
the primary structure of proteins, which includes the amino acid
sequence and post-translational modification (PTM) mapping.[20]
This data, along with immunological characterization data of the
extracted protein, will lead to the identification of a signature pep-
tide. In the future, this signature peptide will be chemically synthe-
sized and utilized as a peptide standard to quantify snow crab TM.
It is well known that a good score in the Basic Local Alignment
Search Tool (BLAST) analysis,[20] together with good separation in
high-performance liquid chromatography (HPLC) analysis, allows
the best choice for the signature peptide.[18,20] The signature
peptide should not have any post-translational modification
(i.e. glycosylation and phosphorylation).[18] The sensitivity and
specificity that MS can provide, with a high-throughput sample
analysisinproteincharacterization,sequencingandPTMmapping,
are ideal for this study.
Purified extract
The acetone powder (10 g) was dissolved in 70 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 overnight at 4 ◦C, followed by centrifugation at 8000 rpm
for 20 min. Total protein was determined in the supernatant
using the Bradford assay, and the supernatant was diluted to
a concentration of 1–2 mg/ml with buffer A. With isoelectric
precipitation (pI 4.6), the proteins were precipitated with 1 M
HCl. The precipitant proteins were redissolved in buffer B (0.20 M
KCl, 25 mM Tris–HCl, pH 8.0, 0.25 M DTT and 0.5 mM EDTA).
TM precipitated with ammonium sulfate 40–70% after the other
proteins have been precipitated in 0–40%. Finally, the pellets were
reconstituted by deionized water, dialyzed against ammonium
bicarbonate by dialysis tubes (MWCO = 12–14 000) overnight at
room temperature and then lyophilized to a powder.
Sodium dodecylsulfate polyacrylamide gel electrophoresis
A pair of 12% SDS-PAGE was used for profiling the purified TM
and the acetone powder extract of snow crab meat from legs and
claw. Protein solution (4–10 µg) was added to each of the wells,
and electrophoresis was run at a voltage of 170 V for 45 min or
until the tracker dye was seen at the base of the gel. One gel was
treated with Coomassie brilliant blue using standard protocol. The
secondgelproteinsweretransferredtoanitrocellulosemembrane
at 100 V for 1 h. After the transfer was completed, the membrane
was placed in a blocking solution (5% skim milk in Tris-buffered
saline (TBS)) for immunoblotting.
This is the first study to analyze the full amino acid sequence
of a TM protein while searching for any protein mutations and
mapping of the sites of PTM and to identify the signature peptide
of snow crab TM using MALDI and ESI-QqToF-MS techniques.
Experimental
Immunoblotting
Chemicals and materials
The rabbit antibody used in this study was generated by exposing
the animals to heat-treated protein extracts from crab, prawn
and lobster using 500 µg of a mixture of these three proteins
sources for each injection. Each protein extract was prepared
from raw crustaceans, the generated protein extract heated to
100 ◦C and the supernatant evaluated by SDS gel electrophoresis
for the presence of TM. Blood samples from the rabbits were
taken at week 0 and 6 to analyze for antibody production, and
All chemicals were used without further purification. Ethanol,
acetone, potassium chloride, ammonium sulfate, acetoni-
trile, hydrochloric acid and methanol were from ACP (Mon-
treal, Canada). Trypsin and endoproteinase (Glu-C V8) se-
quencing grade enzymes were from Promega (Madison, WI,
USA). Tris(hydroxymethyl)aminomethane (Tris), ditheotheritol
(DTT), ethylenediaminetetraacetic acid (EDTA), formic acid
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A. M. Abdel Rahman et al.
the final bleed was conducted at week 9.[21] The immunoblot
was blocked with 5% skim milk solution for 1 h at room
temperature following an overnight incubation (4 ◦C) with the
rabbit antibody (primary antibody) using a working dilution of
1 : 40 000. The blot was washed three times with TBS–Tween. The
secondary antibody used was polyclonal goat anti-rabbit antibody
conjugated to HRP, with a working dilution of 1 : 20 000. The blot
was incubated for 30 min in the secondary antibody solution,
washed again three times with TBS–Tween and incubated with
the substrate TMB. The bands were then visualized using the
enhanced chemiluminescence (ECL) technique.[21]
mixture (1 µl) was deposited onto the first layer and allowed to
dry, which was followed by an on-target wash step. By adding 1 µl
of water on top of the dry spot and blowing the water off using a
pulse of air (after 10 s), the remaining amount of salt was removed.
MALDI-QqToF-MS and CID-MS/MS
MALDI-MS and low-energy collision (CID) analyses were carried
on a QSTAR XL hybrid quadrupole–quadrupole (Qq)/ToF-MS/MS
(Applied Biosystems/MDS Sciex, Foster City, CA, USA) equipped
with an o-MALDI ion source (Applied Biosystems, Foster City, CA,
USA).
To demonstrate the allergenicity of the isolated crab proteins,
different extracts were analyzed for IgE antibody binding from
allergicpatients.Thehumanserawerecollectedfrompatientswith
strong allergic reactivity to shellfish. Ethics approval for this study
was acquired by Monash University as part of an ongoing survey.
For immunoblotting, protein extracts were electrophoretically
separated (see SDS-gels above) and the proteins transferred and
incubated with human serum (diluted 1 : 10 in 1% skim milk)
overnight at 4 ◦C. Subsequently, the blots were washed three
times with PBS-T and membrane-incubated for 1 h in 5 ml of rabbit
antihuman IgE antibody (diluted 1 : 1000) in PBS-T containing 1%
skim milk. After washing the membrane with PBS-T three times, it
was incubated for 30 min in 5 ml of HRP-tagged goat A anti-rabbit
polyclonalantibody(DAKO,Carpentaria,CA,USA)(diluted1 : 1000)
in PBS-T containing 1% skim milk. Finally, the membrane was
washed with PBS three times, and incubated with the substrate
TMB, and the immunoblot membranes were analyzed for IgE
reactivity using the ECL technique.[21]
Electrospray ionization quadruple time-of-flight tandem
mass spectrometry
Peptide separation was conducted using a DIONEX UltiMate3000
Nano LC System (Germering, Germany). A 250-fmol sample of
protein digest dissolved in 0.1% TFA was loaded onto a precolumn
(300 µmi.d.×5 mm,C18 PepMap100,5 µm(LCPacking,Sunnyvale,
CA)) for desalting and concentrating. Peptides were then eluted
from the precolumn and 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%
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 in 55 min, 60–90% in 3 min,
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.
Infurtherexperiments,theESI-MSofthedesalted33-kDaprotein
was directly injected into ESI source, and the resulting multiply
charged spectrum of the protein was deconvoluted by the Analyst
QS 1.1 software.
This protein was further analyzed by CID-MS/MS and the result-
ing peptides spectra were searched by using the National Center
for Biotechnology Information non-redundant (NCBInr) database
with the 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
modificationbecausetheguanidationderivatizationhasbeenper-
formed. Peptides were considered identified if the Mascot score
was over 95% confidence limit.
In-gel digestion and guanidation
The 33-kDa protein bands were excised from the SDS-PAGE plate.
The in-gel guanidation procedure was performed on all protein
samples, which were analyzed by MALDI-QqToF-MS using the
protocol developed by Sergeant et al.[22] This procedure increases
the sensitivity of the lysine-containing peptides by changing the
lysine residue to the homoarginine. The gel pieces were destained
by washing three times with 200 mM of (NH4)2CO3 in solution
of 50% acetonitrile in dH2O, at 30 ◦C for 30 min. The destained
piece was dried under a stream of N2, and then covered by a
solution of 50 mM (NH4)2CO3, pH 7.8, containing 5 ng/µl trypsin
or endoproteinase Glu-C V8 in ice for 30 min for rehydration. After
rehydration, the excess solution was sucked out. The gel was
covered by a solution of 50 mM of (NH4)2CO3 and incubated at
37 ◦C overnight to enhance protein digestion. The water-soluble
peptides were extracted twice with the incubation solution, and
the other remaining peptides were extracted twice with 0.15%
trifluoroacetic acid (TFA) in 50% ACN after a 2-min vortex mixing.
The samples were lyophilized and reconstituted prior to analysis
with 10 µl of 0.1% TFA and desalted with C18 ZipTip.
Results and Discussion
The proteins of snow crab obtained as an acetone powder extract
and as the purified TM extract were profiled by SDS-PAGE, as seen
in Fig. 1(a). The strong band at 33 kDa is presumed to be TM. The
much weaker band >65 kDa, which has a high reactivity against
anti-crustacean polyclonal antibody (Fig. 1(b)) along with patients’
sera (Fig. 1(c) and (d)) that appeared more clearly, was presumed
to either be a dimer or an aggregate form of TM.
The purified protein for both the claw and leg extracts was
further investigated using direct flow injection into ESI source
of the QqToF instrument. The multiply charged ion spectrum of
the purified TM gave a series of multicharged ions, which were
rationalized as [M + 45H]45+ m/z 729.6096; [M + 44H]44+ m/z
Matrix-assisted laser desorption/ionization quadruple time-
of-flight tandem mass spectrometry
Matrix/sample preparation
The two-layer sample/matrix preparation for plate spotting was
employed.[20] The first layer solution consisted 20 mg of HCCA in
1 ml (1/9) methanol/acetone. The second layer solution consisted
40% ACN in H2O saturated by HCCA. The first layer matrix solution
(0.5 µl) was applied to a MALDI target. The second layer matrix
solution (1 µl) was mixed with 1 µl of sample. The sample/matrix
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Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides
Figure 1. Snow crab extract profiling by (a) SDS-PAGE, (b) immunoblotting anti-crustacean polyclonal antibody, (c) immunoblotting patient serum A and
(d) immunoblotting patient serum B, where (1) is claw acetone powder extract, (2) is claw purified TM extract, (3) is leg acetone powder extract and (4) is
leg purified TM extract.
Figure 2. MALDI-QqToF mass spectrum of ‘in-gel’ trypsin digestion of snow crab TM. Each peak in the spectrum represents a tryptic peptide. Their masses
in this spectrum are used for PMF characterization.
746.1454; [M + 43H]43+ m/z 763.2390; [M + 42H]42+ m/z 781.5457;
[M + 41H]41+ m/z 800.7028; [M + 40H]40+ m/z 820.3127; [M +
39H]39+ m/z 841.6777; [M + 38H]38+ m/z 863.9742; [M + 37H]37+
m/z 886.9603; [M + 36H]36+ m/z 912.1664 and [M + 35H]35+ m/z
938.3027. This series of molecular ions was deconvoluted with
the Bayesian protein reconstruct tool, which is available in the
Analyst QS 1.1 software, to give an MW of claw TM extract of
32 783 0.02% Da. Another series of multicharged ions was also
deconvoluted using the same tools to give 32 776 0.02% Da as
the MW of the leg TM extract. The differences in mass between
the calculated and the experimental values are related to the
post-translational modification or to intramolecular modifications
(i.e. N-terminal acetylation (42 Da)).[23]
patients with different symptoms, as seen in Fig. 1(b)–(d). This
permitted the examination of the bioreactivity of the extracted
protein with sera of allergic patients to snow crab TM as well as
the cross-linking reactivity against specific mAb.
The 33-kDa band was excised and one sample was subjected to
in-gel trypsin and another sample to endoproteinase (V8 Glu-C)
digestions. For both enzymatic digestions, the resultant peptides
were extracted and analyzed by MALDI-QqToF-MS (Fig. 2). The
combination of these two enzymes generates two sets of peptides
with different termini and MS sensitivity, which covers more
amino acid motifs during sequencing experiments.[24] The wide
sequencing coverage plays a significant role in the signature
peptide selectivity approach and in the PTM site determination.
The precursor ions of enzymatically digested TM peptide from
both leg and claw extracts were uploaded to the Mascot PMF
search engine. The results were in average score (95) for tryptic
The separated proteins in SDS-PAGE were transferred to the
nitrocellulose membrane and incubated with anti-TM mAb, anti-
crustacean TM polyclonal antibody and sera of two sensitized
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A. M. Abdel Rahman et al.
Figure 3. Representative ESI product ion spectra of selected precursor ions of (a) [M + 2H]2+ = m/z 716.9 and (b) [M + 2H]2+ = m/z 565.3 of two
selected peptides obtained from the tryptic digestion of SC TM having sequences AFANAEGEVAALNR and IVELEEELR, respectively.
c
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J. Mass. Spectrom. 2010, 45, 372–381

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides
Figure 4. ESI product ion spectrum of snow crab TM signature peptide [M + 3H]3+ = m/z 783.4 Da. The axes have been cut to improve peak visualization.
The top (a) and bottom (b) correspond to m/z ranges of 100–800 and 700–1500, respectively.
digestion, which is the top probability based on Mowse scores of
matching withthesnowcrabTMprotein(complementarydeoxyri-
bonucleic acid (cDNA) based Library (NCBInr)). The above scores
are matched with the Mascot algorithms’ criterion: ‘Individual ions
scores >82 indicate identity or extensive homology (p <0.05)’.[25]
For improving the sensitivity of lysine-containing peptides in
the MALDI source and for increasing the sequencing coverage,
the in-gel guanidation reaction was performed for the excised
bands prior to the digestion steps (results are not shown).[21]
Interestingly, both TM samples that were extracted from claws and
legs gave the same amino acid sequence without any indication of
any isoforms as could be deduced from the SDS profile (Fig. 1(a)).
Both MALDI and ESI ionization techniques were used for the
de novo sequencing of the resulting peptides from both types
of protein digestion. In MALDI-CID-MS/MS, the high abundant
peptides’ precursor ions (i.e. 2348.1438, 1432.6904, 1588.8092,
1257.6347,1758.8116,1376.6111,1893.8459,1433.7121,950.4362,
880.4612, 1129.5890, 722.3220, 1789.7741, . . ., etc), some of which
are shown in Fig. 2, were selected in the first quadruple and
fragmented in the low-energy CID collision cell, and the product
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A. M. Abdel Rahman et al.
IntheESI-CID-MS/MSanalyses, thepeptideswerefirstseparated
by nano-HPLC and ionized by a nano-electrospray source. The
data of peptides’ precursor ions and product ions were uploaded
to the Mascot MS/MS ion search engine against the NCBInr
database. The Mowse scores were 746 and 505 as snow crab TM for
the tryptic peptides of TM that has been extracted from claws and
legs, respectively. Table 1 shows the amino acid sequencing and
its distribution between the two enzymatic types. As an example,
two of peptides and their product ion spectra are shown in Fig. 3.
The de novo sequences of some peptides produced from the
sameenzymewereconfirmedbybothMALDI-andESI-CID-MS/MS.
Henceforth, many of the amino acid motifs were confirmed by
Figure 5. Full amino acid sequence of snow crab TM with the calculated
molecular weight 32 655 Da.
ionswerescannedbytheToFanalyzer.Thefullsequencedpeptides
are summarized in the Table 1.
Table 1. De novo sequencing of the product ion spectra obtained from the MALDI-QqToF and ESI-QqToF experiments of the major peptides from
snow crab TM trypsin and V8 digestion
Protease
Trypsin
Peptide sequence
MDAIKKK
Residues #
Missed cleavage
ESI
MALDI
1–7
−
1
1
1
0
1
0
1
0
1
0
0
0
1
0
0
1
0
1
0
−
+
+
+
+
+
+
+
+
−
+
+
+
+
+
+
+
−
+
−
−
−
−
+
+
+
−
+
+
+
−
+
−
+
+
−
−
+
−
+
MQAMKLEK
8–15
DNAMDKADTLEQQNK
AEKTEEEIR
16–30
36–44
SQLVENELDHAQEQLSAATHK
AFANAEGEVAALNRR
IQLLEEDLER
5070
77–91
92–101
92–105
113–125
134–149
141–149
153–160
161–167
168–172
190–198
206–213
218–226
239–244
256–266
269–284
IQLLEEDLERSEER
LAEASQAADESER
SLSDEERMDALENQLK
MDALENQLK
FLAEEADR
KYDEVAR
KLAMVEADLERAEER?
IVELEEELR
SLEVSEEK
EETYKEQIK
AEFAER
LEDELVNEKEK
NIADEMDQAFSELSGF
Endoproteinase Glu-C
KDNAMDKADTLEQQNKE
ANLRAEKTEEE
15–31
32–42
1
3
1
1
1
2
4
1
1
2
2
0
1
5
3
0
2
1
1
0
2
1
+
+
+
+
+
+
−
+
+
+
+
+
+
+
+
+
−
+
+
+
+
+
−
+
+
+
+
−
+
−
−
+
+
−
−
−
−
−
+
−
+
−
+
−
IRANQKKSQLVENE
LDHAQEQLSAATHKLVE
QLSAATHKLVEKE
KEKAFANAEGE
43–56
57–73
63–75
74–84
VAALNRRIQLLEEDLERSEE
RLNTATTKLAEASQAADE
SERMRKVLE
85–104
105–122
123–131
132–145
151–164
165–173
165–177
182–196
188–196
197–208
213–223
224–246
241–252
244–252
253–263
264–273
NRSLSDEERMDALE
ARFLAEEADRKYDE
VARKLAMVE
VARKLAMVEADLE
RAESGESKIVELEEE
SKIVELEEE
LRVVGNNLKSLE
KANQREETYKE
QIKTLANKLKAAEARAE
FAERSVQKLQKE
RSVQKLQKE
VDRLEDELVNE
KEKYKNIADE
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Copyright ꢀ 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 372–381

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides
Table 2. List of putative peptides for phosphorylation as calculated from NetPhos 2.0 with actual mass spectra data reported
NetPhos 2.0
phosphorylation prediction
This study: mass spectrometric data
Predicted
AA residue
Peptide (charge states)
Position
Scorea
m/z
Observed
MW (expt.)
MW (calc.)
Delta
DNAMDKADTLEQQNK (+3)
AEKTEEEIR (+2)
24
39
0.449
0.891
0.046
0.598
0.434
0.936
0.149
0.364
0.569
0.203
0.616
0.988
0.997
0.925
0.996
0.669
0.958
0.943
0.985
0.936
0.056
0.009
0.993
0.646
0.017
–
T
–
T
–
S
–
–
T
–
S
S
S
Y
S
S
S
S
T
Y
–
–
Y
S
–
600.9205
592.7634
810.0429
810.0429
810.0429
613.6226
649.3292
649.3292
649.3292
728.8012
728.8012
653.2906
653.2906
480.7130
892.9011
892.9011
500.7154
500.7154
624.2814
624.2814
645.6856
771.8874
659.3080
1072.9478
1072.9478
574.2832
552.7990
783.4223
783.4223
783.4223
586.9802
609.3691
609.3691
609.3691
688.8408
688.8408
626.6616
626.6616
440.7463
852.9547
852.9547
460.7420
460.7420
584.3200
584.3200
619.0606
731.9433
619.3475
1033.0053
1033.0053
1719.8278
1103.5835
2347.2450
2347.2450
2347.2450
1757.9187
1216.7236
1216.7236
1216.7236
1375.6670
1375.6670
1876.9628
1876.9628
879.4781
1719.7733
1103.5458
2347.1404
2347.1404
2347.1404
1757.8795
1216.6775
1216.6775
1216.6775
1375.6215
1375.6215
1876.8836
1876.8836
879.4450
0.0544
0.0377
0.1046
0.1046
0.1046
0.0392
0.0461
0.0461
0.0461
0.0454
0.0454
0.0792
0.0792
0.0331
0.0735
0.0735
0.0196
0.0196
0.0436
0.0436
0.0915
0.0781
0.0454
0.0816
0.0816
SQLVENELDHAQEQLSAATHK (+3)
SQLVENELDHAQEQLSAATHK (+3)
SQLVENELDHAQEQLSAATHK (+3)
IQLLEEDLERSEER (+3)
RLNTATTKLAE (+2)
50
68
65
102
108
110
111
117
123
134
136
162
185
188
206
210
220
221
227
245
267
279
282
RLNTATTKLAE (+2)
RLNTATTKLAE (+2)
LAEASQAADESER (+2)
LAEASQAADESER (+2)
SLSDEERMDALENQLK (+3)
SLSDEERMDALENQLK (+3)
KYDEVAR (+2)
RAESGESKIVELEEE (+2)
RAESGESKIVELEEE (+2)
SLEVSEEK (+2)
1703.8948
1703.8948
919.4694
1703.8213
1703.8213
919.4498
SLEVSEEK (+2)
919.4694
919.4498
EETYKEQIK (+2)
1166.6255
1166.6255
1854.1601
1461.8720
1236.6804
2063.9961
2063.9961
1166.5819
1166.5819
1854.0686
1461.7939
1236.6350
2063.9146
2063.9146
EETYKEQIK (+2)
QIKTLANKLKAAEARAE (+3)
FAERSVQKLQKE (+2)
KEKYKNIADE (+2)
YKNIADEMDQAFSELSGF (+2)
YKNIADEMDQAFSELSGF (+2)
a The NetPhos 2.0 threshold is >0.500.
either of the ionization sources or by two different digestion
enzymes.
confirmed in a recent study on IgE-binding proteins.[27] However,
the molecular nature of these allergens was not known.
In addition to determining the full sequence of SC TM protein,
another important factor in selecting a signature peptide is the
absence of PTM groups (e.g. phosphorylation and glycosylation).
Consequently, all the precursor mass spectra were evaluated for
the presence of any potential PTM groups. The ESI-MS results
show a MW of 32 783 and 32 776 Da for both claw and leg
extracts, respectively. The experimental MW is compared with
the calculated molecular weight of TM (32 655 Da), which is
based on the cDNA sequence,[28] and the difference in mass
indicatesthepresenceofacetylatedN-terminusandsomevariable
modifications (i.e. oxidation of methionine). This was confirmed by
the theoretical algorithm of the NetAcet 1.0 server tool,[29] which
indicated that the only site applicable for acetylation was the
N-terminal methionine. This type of modification is quite common
for the Eukaryotic TM, as reported for the bovine, chicken (P04268)
and human (P09493) cases in the UniProtKB/Swiss-Prot data bases.
The precursor ion and intensity data generated from PMF
experiments were uploaded on the ExPASy FindMod tool,
to check if there was any potential peptide having any PTM
motif(s). The obtained report indicated the absence of any
type of modifications. Further confirmation of the absence of
modified peptide ions was undertaken by usually searching for
the calculated molecular ions of PTM motifs in precursor spectra.
The possible sites of phosphorylation of SC TM were identified
using the NetPhos 2.0 server,[30] which can reveal the T, S and
Analyzing the results in Table 1, it is interesting to note that
the N-terminus peptide (residues 1–7, with sequence MDAIKKK)
as a major ion was difficult to observe (ionize) and to produce a
product spectrum. This is an indication that it was blocked with
the acetyl group at the N-terminus, which is commonly found in
the Eukaryotic TM (detailed below).
The size difference between trypsin and V8 enzymes limits the
V8 enzyme to penetrate far inside the gel pores. This can lead to
reduction in in-gel digestion efficiency. Many of the V8 resultant
peptides are identified with many missed cleavages up to five,
whereas the tryptic peptides were with a maximum of one missed
cleavage (Table 1).
In addition, a study was undertaken to identify the chemical na-
tureofthehighermolecularbands, whichhavestrongantibodyre-
activitytobothrabbitandpatientsIgE.The65-kDabandoftheSDS-
PAGE was excised, destained and digested using the same proto-
cols described above. Remarkably, the same amino acid sequence
was obtained by the de novo sequencing, but with higher relative
intensities of the peptide signals. This is the first study to conclu-
sively demonstrate that the higher molecular IgE antibody binding
protein is a dimer of the allergen TM. This is of significant impor-
tance, asseveralaerosolizedallergenicproteinshavebeendemon-
stratedinthecrustaceanprocessingindustrysincethediscoveryof
occupational asthma in the snow crab processing industry[26] and
c
J. Mass. Spectrom. 2010, 45, 372–381
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms

A. M. Abdel Rahman et al.
Y motifs of major phosphorylated amino acids in Eukaryotic TM
protein (Table 2). The scores of predicted sites, positions and
molecular ions of the peptides that possess these phosphorylated
sites are estimated and then compared with the present (ESI or
MALDI) precursor spectra. Therefore, this evaluation suggests
that there was no match between the predicted phosphorylated
molecular ions and the precursor experimental molecular ion in
PMF analyses. This result was further confirmed by the lack of any
phosphorylated immonium ions on the product ion spectra.
All the resultant peptides produced by both trypsin and
V8 enzymes were introduced to the BLAST test to find out
which of the peptides is the best candidate as a signature
peptide to be used as a TM chemical surrogate in future
quantitative work in the environmental researches. The NCBI
BLAST test, which is used to find regions of local similarity
between sequences of the NCBI database and calculates the
statistical significance of matches, reported that the peptide
located at 50–70 (SQLVENELDHAQEQLSAATHK) is an ideal
signature peptide (Figs 4 and 5) for TM (Chionoecetes opilio)
with 100% identity, score = 68.5 bits (154), and expect. =
6e−11. This peptide also got the best scores as homology and
identity by the Mascot search engine.[25] Therefore the chosen
signature peptide for future quantitation development is shown
below:
Health & Safety Research, Memorial University of Newfound-
land. We would like to acknowledge Dr Barbara Neis (SafstyNet),
Memorial University of Newfoundland, and Department of Chem-
istry and NSERC for the financial support. Finally, the Genomic
and Proteomics Facility and D. Jackman (Biochemistry) are
acknowledged.
References
[1] A. L. Lopata, S. B. Lehrer. New insights into seafood allergy. Curr.
Opin. Allergy Clin. Immunol. 2009, 9, 270.
[2] Food and Agriculture Organization of the United Nations (FAO).
The State of World Fisheries and Aquaculture 2006. Fisheries and
Aquaculture Department: Rome, 2007.
[3] M. F. Jeebhay, T. G. Robins, M. E. Miller, E. Bateman, M. Smuts,
R. Baatjies, A. L. Lopata. Occupational allergy and asthma among
salt water fish processing workers. Am. J. Ind. Med. 2008, 51, 899.
[4] M. F. Jeebhay, T. G. Robins, S. B. Lehrer, A. L. Lopata. Occupational
seafood allergy – a review. Occup. Env. Med. 2001, 58, 553.
[5] Y. Lu, T. Ohshima, H. Ushio, Y. Hamada, K. Shiomi. Immuological
characteristics of monoclonal antibodies against shellfish major
allergen tropomyosin. Food Chem. 2007, 100, 1093.
[6] D. R. Hoffman, E. D. Day, J. S. Miller. The major heat stable allergen
of shrimp. Ann. Allergy 1981, 47, 17.
[7] Y. L. Liang, M. J. Cao, W. J. Su, L. J. Zhang, Y. Y. Huang, G. M. Liu.
Identification and characterization of the major allergen of Chinese
mitten crab (Eriocheir sinensis). Food Chem. 2008, 111, 998.
[8] M. F. Jeebahay, T. G. Robins, N. Seixas, R. Baatjies, D. A. George,
E. Rusford, S. B. Lehrer, A. L. Lopata. Environmental exposure
characterization of fish processing workers. Ann. Occup. Hyg. 2005,
49, 423.
SQLVENELDHAQEQLSAATHK
[9] J. L. Malo, P. Chretien, M. Mccants, S. B. Lehrer. Detection of Snow
crab antigens by air sampling of a snow crab production plant. Clin.
Exp. Allergy 1997, 27, 75.
Conclusion
[10] B. M. Wolska, D. F. Wieczorek. The role of tropomyosin in the
regulation of myocardial contraction and relaxation. Pflugers Arch.
2003, 446, 1.
It was demonstrated for the first time that the full length of a major
crustacean allergen, TM, can be sequenced utilizing a combination
of ESI and MALDI using a hybrid QqToF-MS (Fig. 5).
[11] W. G. Lewis, L. B. Smillie. The amino acid sequence of rabbit cardiac
Using a ‘bottom-up’ MS approach,[31]the sequence of TM from
legs and claws of the snow crab (C. opilio) demonstrated to have
the same amino acid sequence and an average molecular weight
of 32 733 Da, which is slightly higher than previously reported.
Using precursor ion spectra, it was determined that N-terminal
acetylation is the site for post-translational modification, this
accounting for the small MW difference (stated above). In addition,
a higher MW protein, which is immunologically recognized by
patient sera, was identified as the TM dimer. This finding is
of significant importance, as previous studies on environmental
allergen exposure among snow crab processors were not able
to identify the nature of the generated protein allergens during
processing. Basedonmoleculardataobtained, asignaturepeptide
of 21 amino acids was chosen to surrogate chemically the
full-length SC TM. The identification of a signature peptide
for the allergen TM using MALDI-QqToF-MS is fundamental
for the development of sensitive and specific quantification
methods of this highly allergenic protein in the work place.
Furthermore, the results of this study clearly demonstrate the
strength of mass spectroscopy in characterizing minute amounts
of unknown allergens, which will be vital in the identification of
yet unknown snow crab allergens responsible for occupational
sensitization.
tropomyosin. J. Biol. Chem. 1980, 255, 6854.
[12] D. H. Heeley, T. Bieger, D. M. Waddleton, C. Hong, D. M. Jackman,
C. McGowan, W. S. Davidson, R. C. Beavis. Characterisation of fast,
slow and cardiac muscle tropomyosins from salmonid fish. Eur. J.
Biochem. 1995, 232, 226.
[13] G. Reese, S. Schicktanz, I. Lauer, S. Randow, D. Luttkopf, L. Vogel,
S. B. Lehrer, S. Vieths. Structural, immunological and functional
properties of natural recombinant Pen a 1, the major allergen
of Brown Shrimp, Penaeus aztecus. Clin. Exp. Allergy. 2006, 36, 517.
[14] M. Geng, J. Ji, F. E. Regnier. Signature-peptide approach to
detecting protein in complex mixtures. J. Chromatogr. A 2000,
870, 295.
[15] L. Kluwe, K. Maeda, A. Miegel, S. Fujita-Becker, Y. Maeda, G. Talbo,
T. Houthaeve, R. Kellner. Rabbit skeletal muscle αα-tropomyosin
expressed in baculovirus-infected insect cells possessed the
authentic N-terminus structure and function. J. Muscle Res. Cell
Motil. 1995, 16, 103.
[16] M. Rosmilah, M. Shahnaz, A. Masita, A. Noormalin, M. Jamaludin.
Identification of major allergens of two species of local snappers:
Lutjanusargentimaculatus(merah/redsnapper)andLutjanusjohnii
(jenahak/golden snapper). Trop. Biomed. 2005, 22, 171.
[17] A. R. Lorenz, S. Scheurer, D. Haustein, S. Vieths. Recombinant food
allergens. J. Chromatogr. B 2001, 756, 255.
[18] A. M. Cohen, A. A. Mansour, J. H. Banoub. ‘De novo’ sequencing
of Atlantic cod vitellogenin tryptic peptides by matrix-assisted
laser desorption/ionization quadrupole time-of-flight tandem mass
spectrometry: similarities with haddock vitellognin. RapidCommun.
Mass Spectrom. 2005, 19, 2454.
[19] J. H. Banoub, A. M. Cohen, A. A. Mansour, P. Thibault. Characteri-
sation and de novo sequencing of Atlantic salmon Vitellogenin
protein by electrospray tandem and matrix-assisted laser desorp-
tion/ionization mass spectrometry. Eur. J. Mass Spectrom. 2004, 10,
121.
Acknowledgements
This research was partially funded by a Canadian Institutes of
Health Research Interdisciplinary Capacity Enhancement Grant
(ICH 62328) through the SafetyNet Centre for Occupational
[20] M. Kinter, N. E. Sherman. Protein Sequencing and Identification Using
Tandem Mass Spectrometry. John Wiley: New York, 2000.
c
www.interscience.wiley.com/journal/jms
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
J. Mass. Spectrom. 2010, 45, 372–381

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides
[21] A. L. Lopata, M. F. Jeebhay, G. Reese, J. Fernandes, B. Fenemore,
A. Elliott, T. G. Robins, S. B. Lehrer. Detection of fish antigens
aerosolized during fish processing using newly developed
immunoassays. Int. Arch. Allergy 2005, 138, 21.
[27] B. V. Gill, T. R. Rice, A. Cartier, D. Gautrin, B. Neis, L. Horth-Susin,
M. Jong, M. Swanson, S. B. Lehrer. Identification of crab proteins
that elicit IgE reactivity in snow crab – processing workers. J.Allergy.
Clin. Immunol. 2009, 124, 1055.
[22] K. Sergeant, B. Samyn, G. Debyser, J. V. Beeumen. ‘De novo’
sequence analysis of N-terminal sulfonated peptides after in gel
quanidination. Proteomics 2005, 5, 2369.
[23] M. C. Huang, Y. Ochiai. Fish fast skeletal muscle tropomyosins show
species-specific thermal stability. Comp. Biochem. Physiol. B 2005,
141, 461.
[28] K. Motoyama, Y. Suma, S. Ishizaki, Y. Nagashima, K. Shiomi.
Molecular cloning of tropomyosins identified as allergens in six
species of crustaceans. J. Agric. Food Chem. 2007, 55, 985.
[29] L. Kiemer, J. D. Bendtsen, N. Blom. NetAcet: Prediction of N-terminal
acetylation sites. Bioinformatics 2005, 21, 1269.
[30] N. Blom, S. Gammeltoft, S. Brunak. Sequence and structure-based
prediction of Eukaryotic protein phosphorylation sites. J. Mol. Biol.
1999, 294, 1351.
[24] E. Hoffmann, V. Stroobant. Mass Spectrometry: Principles and
Applications. John Wiley: New York, 2007.
[25] D. J. Pappin, P. Hojrup, A. J. Bleasby. Rapid identification of proteins
by peptide-mass fingerprinting. Curr. Biol. 1993, 3, 487.
[26] A. Cartier, J. L. Malo, F. Forest, M. Lafrance, L. Pineau, J. J. St-Aubin,
J. Y. Dubois. Occupationalasthmainsnowcrab-processingworkers.
J. Allergy Clin. Immunol. 1984, 74, 261.
[31] A. M. Cohen, F. Jahouh, S. Sioud, R. M. Rideout, M. J. Morgan,
J. H. Banoub. Quantification of Greenland halibut serum
vitellogenin: a trip from the deep sea to the mass spectrometer.
Rapid Commun. Mass Spectrom. 2009, 23, 1049.
c
J. Mass. Spectrom. 2010, 45, 372–381
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/jms