Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry
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  This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:  Author's personal copy Biomolecular characterization of allergenic proteins in snow crab (  Chionoecetes opilio  ) and  de novo  sequencing of the secondallergen arginine kinase using tandem mass spectrometry  Anas M. Abdel Rahman a, ⁎  , Sandip D. Kamath b , Andreas L. Lopata b,c  , John J. Robinson d , Robert J. Helleur  a a Department of Chemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, A1B 3X7, Canada b School of Applied Science, Allergy Research Group, RMIT University, Bundoora Campus Melbourne, Bundoora 3083 Victoria, Australia c Comparative Genomics Centre,Molecular Sciences Bldg 21, James Cook University, Townsville, 4811, Queensland, Australia d Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, A1B 3X9 Canada A R T I C L E I N F O A B S T R A C T  Article history: Received 27 August 2010Accepted 28 October 2010Snow crab ( Chionoecetes opilio ) proteins have been recognized as an important source of bothfood and occupational allergens. While snow crab causes a significant occupational allergy,only one novel allergen has recently been fully characterized. The muscle proteins fromsnow crab legs were profiled by SDS-PAGE. Several of these proteins were characterizedusing tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20 kDa), arginine kinase (40), troponin (23 kDa) and  α -actine (42 kDa) and smoothendoplasmic reticulum Ca 2+ ATPase (113 kDa).Immunoblottingusingserum ofsixteen allergic patientsresultedinstrongreactivitywiththe40-kDa protein in seven patients (43%). This protein was purified by chromatography andsubsequently  de novo  sequenced using matrix assisted laser desorption ionization andelectrospray tandem mass spectrometry. Weidentified a second important allergen, argininekinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for argininekinase. The suitability of this signature peptide was tested for analytically representing thearginine kinase, by performing a multi-reaction monitoring tandem mass spectrometryapproach on actual air filter samples collected from a simulated crab processing plant.© 2010 Elsevier B.V. All rights reserved. Keywords: Snow crab ( Chionoecetes opilio )Arginine kinase De novo  sequencing Mass spectrometryAllergenQuantification 1. Introduction The snow crab (SC) fishery in Atlantic Canada represents theworld's largest SC fishery; it accounts for almost 90% of worldlandings (by weight) in 2004, with over half of these landingscoming from Newfoundland and Labrador. In 2007, the NLfishery alone landed 50,000 t, valued at $177 million [1].Unfortunately, SC meat can be one of the most importantcauses of severe acute hypersensitivity reactions, including fatalanaphylaxisandsevereasthmaamongthefishermenandprocessing plant workers [2]. Fish and shellfish are a leading  causeofIgE-mediatedfoodhypersensitivity[2 – 4].IgE-mediatedreactions that cause nausea, vomiting, abdominal pain, anddiarrhea may be triggered within minutes of ingestion [5].The molecular structure of tropomyosin, the major allergenin crustaceans [5 – 7], was recently characterized in snow craband black tiger prawn using mass spectrometry [8,9]. Besides tropomyosin, other allergenic proteins from crustaceans have  J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 3 1  –  2 4 1 ⁎  Corresponding author.  Tel.: +1 709 864 2181.E-mail address: (A.M. Abdel Rahman).1874-3919/$  –  see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.jprot.2010.10.010 available at  Author's personal copy been reported. In 2003, Yu et al. [10] identified a novel shrimpallergen designated as Pen m 2 from ( Penaeus monodon ) by two-dimensional immunoblotting using sera from shrimp allergicpatients. The allergen was identified using cDNA cloning. Theopenreadingframeencoded356aminoacidswithatheoreticalmolecular weight of ~40kDa. The amino acid sequence of thisprotein showed 60% similarity to arginine kinase of thecrustacean, Kuruma prawn ( Penaeus japonicus ) [10] . Argininekinase(AK)wasrecentlyreportedasanallergenindifferent crustacean and invertebrate species. AK was identi-fied in white shrimp ( Litopenaeus vannamei ) [11] ,  gulf shrimp( Penaeusaztecus )[12] , Chineseshrimp( Fenneropenaeuschinensis )[13] ,  and other shrimp species using a proteomics approach[14]. Moreover, AK has been identified in other invertebratessuch as the house dust mite ( Dermatophagoides farinae ) [15] , Indian-meal moth ( Plodia interpunctella ) [16] ,  silkworm larvae( Bombyx mori ) [17] ,  and American cockroach ( Periplanetaamericana ) [18 – 20] . Monitoring airborne allergens in SC harvesting and proces-sing workplaces is essential to reducing the worker's risk of developingallergenicairwaydiseases[21].Normally,allergensare characterized and measured through immunologicalreactivity by enzyme-linked immunosorbent assay (ELISA)[22 – 24], radioallergosorbent rest (RAST) [25], and immunoblot- ting  [22 – 26]. These techniques evaluate the total proteinconcentration which includes non-allergenic proteins inaddition to non-SC allergens [27].Quantifying the major SC allergens as a way of correlating their amounts with the severity of the allergen exposure andin determining the threshold values requires a highlysensitive, specific, and reproducible technique. Isotopic dilu-tion mass spectrometry has played a crucial role in proteinquantification in the last two decades, provoking the use of this technique for the present study [27].Snow crab tropomyosin was previously characterized and de novo  sequenced [8]. Arginine kinase is also a protein of interest since it has been identified in other crustaceans butnot snow crab [11 – 14].In this study snow crab crude extract was profiled on SDS-PAGE and screened against patients ’  sera. Different allergenicproteins from this profile were characterized using tandemmass spectrometry. Previously, we identified and character-ized the major allergen tropomyosin [8,9]. Arginine kinase, which is one of the most immunoactive protein was isolatedand purified from the crude extract. The allergenicity of purified AK was examined by immunoblotting with allergenicpatients ’  sera. The tryptic peptides were generated along withdifferent types of derivatization reactions used in amino acidsequencing. The most abundant peptides were characterizedusing both ESI and MALDI ion sources, for protein identifica-tionand de novo sequencingusingpeptidemassfingerprinting (PMF) and peptide fragment fingerprinting (PFF), respectively.The mass spectra data were uploaded to the Mascot databasesearch engine. The AK homology between snow crab andorange mud crab, which is the closest species, was studied.The active site motifs were evaluated using selected bioinfor-matics algorithms to confirm a signature pattern for AK. Aunique and abundant tryptic peptide was selected andevaluated as a chemical surrogate for AK. Finally, thissignature peptide and its deuterated isotopic homolog using d 3 - L -alanine-were chemically synthesized and used in apreliminary study to develop a sensitive and specific quanti-fication method for AK using multi-reaction monitoring (MRM) LC – MS/MS. The levels of the snow crab AK, for thefirsttime,weremonitoredinactualairsamplescollectedfroma simulated processing plant. A tryptic-digested snow crabcrude extract sample was used as a positive control. 2. Experimental 2.1. Chemicals and materials All chemicals wereusedwithout further purification. Ammoni-um sulfate, acetonitrile, hydrochloric acid, and methanol weresupplied by ACP (Montreal, Canada). Trypsin sequencing gradeenzymes were purchased from Promega (WI, USA). Tris(hydro-xymethyl)aminomethane (Tris), dithiotheritol (DTT), ethylene-diaminetetraacetic acid (EDTA), formic acid (FA), ammoniumbicarbonate,o-methylisoureahemisulfate,ammoniumhydrox-ide,horseradishperoxidase(HRP),Chemiluminescentsubstrate,Sodium dodecylsulphate (SDS), ammonium formate and  α -cyano-4-hydroxycinamic acid (HCCA) matrix were purchasedfromSigma-Aldrich (St. Louis, MO, USA). TheBradfordassay kitand PVDF immunoblot membrane were from BioRad (Hercules,CA, USA). The dialysis bags were purchased from Fischer Scientific (Roncho Dominguez, CA, USA). For desalting, theZipTip C 18  filters were purchased from Millipore Corporation(Bedford, MA, USA). The skimmed milk powder was purchasedfrom the local supermarket. Tris Buffered Saline (TBS) andPhosphate Buffered Saline (PBS) tablets were purchased fromAmresco, USA. The photo-sensitive films were purchased fromAmersham Bioscience, Germany. The developer and fixer werepurchased from Kodak, USA. RapiGest SF surfactant fromWaters Corporation (Milford, MA, USA), TopTip filters withPoly-Hydroxyethyl(HILIC)resinfromPolyLCInc.(Columbia,MD,USA), and 33-mm polytetrafluoroethylene (PTFE) filters for air sampling was bought from SKC Inc. (Eighty Four, PA, USA). Thesignature peptide,  LVSAVNEIEK  (purity>98.33%; molar mass=1101.27Da) and its deuterated isotopic homolog using d 3 - L -alanine (purity>96.80%; molar mass 1104.27 Da) werepurchased from GeneMed Synthesis (San Francisco, CA, USA). 2.2. Arginine kinase purification and SDS profiling Arginine kinase was purified from crab leg muscle extract byammoniumsulfateprecipitationfollowingaprotocoldevelopedby Garcia-Orozco et al. [11].ThetotalproteinconcentrationofthecrudeextractandtheAK isolate was determined using the Bradford assay. Sincethiscrudeextractdoesnotcontainmanydifferentproteinsweutilized a 12% SDS-PAGE gel electrophoresis for proteinprofiling of the crude extract, the ammonium sulfate precip-itated fractions, and the purified AK. A protein solution (10  μ g)wasaddedtoeachofthewells,andelectrophoresiswasrun at170 V for 45 min, or until the tracker dye was seen at the baseofthegel.OnegelwastreatedwithCoomassieBrilliantBlueR-250,usingstandard protocol.For thesecondgel,proteinsweretransferred to a PVDF membrane at 15 V for 20 min. After thetransfer was completed, the membrane was placed in a 232  J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 3 1  –  2 4 1  Author's personal copy blocking solution (5% skimmed milk in Tris-buffered saline(TBS)) for immunoblotting. 2.3. Immunoblotting To demonstrate the allergenicity of the isolated crab proteins,different bands were analyzed for IgE antibody binding fromallergic patients with marked allergic symptoms to shellfish.Patients for this study were selected based on clinicalreactivity to shellfish. Ethics approval for this study wasacquired by Monash University as part of an ongoing survey.For immunoblotting, protein extracts were electrophoreticallyseparated (see SDS gels above), proteins transferred andincubated with human serum (diluted 1:10 in 1% skimmedmilk) overnight at 4 °C using a slot-blot apparatus (IdeaScientific, USA). Subsequently, blots were washed threetimes with PBS-T and the membrane incubated for 1 h in5 mL of HRP labeled rabbit polyclonal antihuman IgE antibody(DAKO, USA) (diluted 1:2000) in PBS-T containing 1% skimmedmilk.Afterwashingthemembrane withPBS-Tthreetimestheblots were incubated with the chemiluminescent substrateand analyzed for IgE reactivity using ECL technique [8,22]. 2.4.  In-gel  digestion and guanidation Six immunoreactive proteins, as identified by SDS-gel elec-trophoresis and subsequent immunoblotting with allergicpatient serum, were excised from SDS-PAGE gels. An in-gelguanidation procedure was performed on these selectedproteins for subsequent MALDI-QqToF-MS using the protocoldeveloped by Sergeant et al. [28]. This procedure increases thesensitivity of the lysine-containing peptides by changing thelysine residue to the homoarginine. The gel pieces weredestainedbywashingthreetimeswith200 mMof(NH 4 ) 2 CO 3 insolution of 50% acetonitrile in dH 2 O, at 30 °C for 30 min. Thedestained piece was dried under a stream of N 2 , and thencovered by a solution of 50 mM (NH 4 ) 2 CO 3  pH 7.8, containing 5 ng/ μ L trypsin in ice for 30 min for rehydration. After rehydration, the excess solution was sucked out. The gel wascovered by a solution of 50 mM of (NH 4 ) 2 CO 3  and incubated at37 °C overnight to enhance protein digestion. The water soluble peptides were extracted twice with the incubationsolution and other remaining peptides extracted twice with0.15%TFA in 50% ACN after a 2 min vortex mixing. Thesamples were lyophilized, and reconstituted, prior to analysiswith 10  μ L of 0.1% TFA and desalted with C 18  ZipTip. 2.5. MALDI-QqToF-MS 2.5.1. 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 of H 2 O saturated by HCCA.0.5  μ L of the first layer matrix solution was applied to aMALDI target. 1  μ L of the second layer matrix solution wasmixed with 1  μ L of sample. 1  μ L of the sample/matrixmixture was deposited onto the first layer and allowed todry, followed by an on-target wash step. By adding 1  μ L of water on top of the dry spot and blowing the water off using a pulse of air (after 10 s), a subsequent amount of salt wasremoved. 2.6. MALDI-QqToF-MS and CID-MS/MS MALDI-MS and low energy collision (CID) analyses werecarried out on a QSTAR XL hybrid quadrupole – quadrupole/time-of-flight tandem mass spectrometer (QqToF-MS/MS)(Applied Biosystems/MDS Sciex, Foster City, USA) equippedwith an o-MALDI ion source (Applied Biosystems, Foster City, CA). 2.7. LC-ESI-QqToF-MS Peptide separation was conducted using a DIONEX Ulti-Mate3000 Nano LC System (Germering, Germany). A 250 fmolsample of protein digest dissolved in 0.1% TFA was loadedonto a pre-column (300  μ m ID×5 mm, C 18  PepMap 100, 5  μ m(LC Packing, Sunnyvale, CA) for desalting and concentrating.Peptidesweretheneluted fromthepre-columnandseparatedon a nanoflow analytical column (75  μ m ID×15 cm, C18PepMap 100, 3  μ m, 100 A, (LC Packing, Sunnyvale, CA) at180 nL/min using the following gradient. The aqueous mobilephases consisted of (A) 0.1% formic acid/0.01% trifloroaceticeacid/2% ACN and (B) 0.08% formic acid/0.008% trifloroaceticacid/98% ACN. A gradient of 0% B for 10 min, 0 – 60% B in55 min, 60 – 90% in 3 min, and 90% B in 5 min was applied.Including a regeneration step one run was 106 min long.The ESI-MS of the LC-eluting peptides were measured withthe same hybrid QqToF-MS/MS system equipped with ananoelectrospray source (Protana XYZ manipulator). Thenanoelectrospray was generated from a PicoTip needle (10  μ mi.d., New Objectives, Wobum, USA) at a voltage of 2400 V.This protein was further analyzed by CID-MS/MS and theresulting peptides spectra were searched by using the NationalCenter for Biotechnology Information non-redundant database(NCBInr) 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 modificationandguanidinyl(K)asafixedmodificationsincetheguanidationderivatisation has been performed. Peptides were consideredidentified if the Mascot score was over 95% confidence limit. 2.8. Air sample collection, protein extraction andtryptic digestion Airborne snow crab proteins were generated by processing crab in a simulated crab processing station and air sampleswere collected as described in a previous study that measuredairborne crab tropomysin [27]. The procedures for air samplecollection, protein extraction from filters, tryptic digestion,and detergent removal prior to LC – MS/MS analysis wereperformed as described elsewhere [27]. 2.9. Multi reaction monitoring LC  –  MS/MSquantification method Signature peptide analysis was performed following the MRMLC – MS/MS protocol developed by Abdel Rahman et al. [27]with the exception of the mass spectrometric conditions: the 233  J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 3 1  –  2 4 1  Author's personal copy eluted peptides ’  droplets were desolvated at the ESI with gasflowrateof400 L/handatemperatureof250 °C.Theionswereaccelerated through the capillary and orifice cone at 3.02 kVand 40 V, respectively. The precursor ions were fragmented atlow energy CID using argon gas and the collision energy of 13 eV. The [M+2 H]  2+ precursor ions of the unlabeled and Table 1  –  List of the allergenic muscle proteins from snow crab and the  de novo  sequences of their relevant peptides. Protein name Molecular mass (kDa) Mascot score Selected peptides Sarcoplasmic Ca-binding 20 44  108 VATVSLPR 115 Troponin 23 118  38 KGFMTPER 45162 AAEFNFR 168 Tropomyosin 33 494 Ref. [8]Arginine kinase 40 885 Table 2 α -Actine 42 891  21 AGFAGDDAPR 3031 AVFPSIVGRPR 4153 DAYVGDEAQSKR 6464 RGILTLK 7098 IAPEESPVLLTEAPLNPK  115150 TTGIVLDTGDGVTHTVPIYEGYCLPHAILR 179180 LDLAGRDLTAYLTK 193199 GYSFTTTAEREIVR 212241 SYELPDGQVITIGNER 256287 CDIDIRK 293293 KDLFANNVLSGGTTMYPGIADR 314318 EITALAPPTIK 328329 IKIIAPPER 337331 IIAPPERK 338362 EEYDESGPGIVHR 374 SERCA/smooth endoplasmicreticulum Ca 2+ ATPase113 585  36 YGPNELPAEEGK 47111 NAESAIEALKEYEPEMGK 128144 EIVPGDLVEISVGDKIPADLR 164175 IDQSILTGESVSVIK 189206 NILFSGTNVAAGK 18237 TQMAETEEIKTPLQQK 252437 VGEATETALIVLGEK 451482 EFTLEFSR 489621 VIVITGDNK 629713 KAEIGIAMGSGTAVAK 728 Fig. 1  –  (A) Crude extract of snow crab muscle protein analyzed by SDS-PAGE Coomassie staining and characterized byimmunoblotting using a pool of patients ’  IgE antibody. (B) Purified arginine kinase analyzed by SDS-PAGE Coomassie staining,and by immunoblot IgE antibody binding using serum from seven patients. 234   J O U R N A L O F P R O T E O M I C S 7 4 ( 2 0 1 1 ) 2 3 1  –  2 4 1
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