Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry
Graphical abstract
Introduction
The snow crab (SC) fishery in Atlantic Canada represents the world's largest SC fishery; it accounts for almost 90% of world landings (by weight) in 2004, with over half of these landings coming from Newfoundland and Labrador. In 2007, the NL fishery alone landed 50,000 t, valued at $177 million [1]. Unfortunately, SC meat can be one of the most important causes of severe acute hypersensitivity reactions, including fatal anaphylaxis and severe asthma among the fishermen and processing plant workers [2]. Fish and shellfish are a leading cause of IgE-mediated food hypersensitivity [2], [3], [4]. IgE-mediated reactions that cause nausea, vomiting, abdominal pain, and diarrhea may be triggered within minutes of ingestion [5].
The molecular structure of tropomyosin, the major allergen in crustaceans [5], [6], [7], was recently characterized in snow crab and black tiger prawn using mass spectrometry [8], [9]. Besides tropomyosin, other allergenic proteins from crustaceans have been reported. In 2003, Yu et al. [10] identified a novel shrimp allergen designated as Pen m 2 from (Penaeus monodon) by two-dimensional immunoblotting using sera from shrimp allergic patients. The allergen was identified using cDNA cloning. The open reading frame encoded 356 amino acids with a theoretical molecular weight of ~ 40 kDa. The amino acid sequence of this protein showed 60% similarity to arginine kinase of the crustacean, Kuruma prawn (Penaeus japonicus) [10].
Arginine kinase (AK) was recently reported as an allergen in different crustacean and invertebrate species. AK was identified in white shrimp (Litopenaeus vannamei) [11], gulf shrimp (Penaeus aztecus) [12], Chinese shrimp (Fenneropenaeus chinensis) [13], and other shrimp species using a proteomics approach [14]. Moreover, AK has been identified in other invertebrates such as the house dust mite (Dermatophagoides farinae) [15], Indian-meal moth (Plodia interpunctella) [16], silkworm larvae (Bombyx mori) [17], and American cockroach (Periplaneta americana) [18], [19], [20].
Monitoring airborne allergens in SC harvesting and processing workplaces is essential to reducing the worker's risk of developing allergenic airway diseases [21]. Normally, allergens are characterized and measured through immunological reactivity by enzyme-linked immunosorbent assay (ELISA) [22], [23], [24], radioallergosorbent rest (RAST) [25], and immunoblotting [22], [23], [24], [25], [26]. These techniques evaluate the total protein concentration which includes non-allergenic proteins in addition to non-SC allergens [27].
Quantifying the major SC allergens as a way of correlating their amounts with the severity of the allergen exposure and in determining the threshold values requires a highly sensitive, specific, and reproducible technique. Isotopic dilution mass spectrometry has played a crucial role in protein quantification 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 but not snow crab [11], [12], [13], [14].
In this study snow crab crude extract was profiled on SDS-PAGE and screened against patients’ sera. Different allergenic proteins from this profile were characterized using tandem mass spectrometry. Previously, we identified and characterized the major allergen tropomyosin [8], [9]. Arginine kinase, which is one of the most immunoactive protein was isolated and purified from the crude extract. The allergenicity of purified AK was examined by immunoblotting with allergenic patients’ sera. The tryptic peptides were generated along with different types of derivatization reactions used in amino acid sequencing. The most abundant peptides were characterized using both ESI and MALDI ion sources, for protein identification and de novo sequencing using peptide mass fingerprinting (PMF) and peptide fragment fingerprinting (PFF), respectively. The mass spectra data were uploaded to the Mascot database search engine. The AK homology between snow crab and orange mud crab, which is the closest species, was studied. The active site motifs were evaluated using selected bioinformatics algorithms to confirm a signature pattern for AK. A unique and abundant tryptic peptide was selected and evaluated as a chemical surrogate for AK. Finally, this signature peptide and its deuterated isotopic homolog using d3-L-alanine-were chemically synthesized and used in a preliminary study to develop a sensitive and specific quantification method for AK using multi-reaction monitoring (MRM) LC–MS/MS. The levels of the snow crab AK, for the first time, were monitored in actual air samples collected from a simulated processing plant. A tryptic-digested snow crab crude extract sample was used as a positive control.
Section snippets
Chemicals and materials
All chemicals were used without further purification. Ammonium sulfate, acetonitrile, hydrochloric acid, and methanol were supplied by ACP (Montreal, Canada). Trypsin sequencing grade enzymes were purchased from Promega (WI, USA). Tris(hydroxymethyl)aminomethane (Tris), dithiotheritol (DTT), ethylenediaminetetraacetic acid (EDTA), formic acid (FA), ammonium bicarbonate, o-methylisourea hemisulfate, ammonium hydroxide, horseradish peroxidase (HRP), Chemiluminescent substrate, Sodium
Result and discussion
The crude extract of the SC proteins (from leg sections) was profiled by SDS-PAGE as shown in Fig. 1A. Proteins from numerous bands were excised and treated with tryptic digestion for further characterization by bottom up tandem mass spectrometry approach. Several protein bands within the range 20–113 kDa were analyzed and their relevant peptides de novo sequenced using PFF by tandem mass spectrometry. The precursor and product spectra of the peptides were uploaded and searched in Mascot search
Conclusion
Arginine kinase has recently been reported as a major seafood allergen in prawns. The present study evaluates, for the first time, the allergenicity of the snow crab AK, by examining its immunoreactivity with the sera of seven allergic patients. The primary structure of the AK was characterized through amino acid sequencing using bottom up tandem mass spectrometry. The amino acid sequence was registered in the UniProt knowledgebase. The active site of AK was determined with a consensus pattern
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 Health & Safety Research, Memorial University of Newfoundland and the Asthma Foundation Victoria (AL). We would like to acknowledge Dr. Barbara Neis (SafetyNet), Memorial University of Newfoundland (NSERC) and the Department of Chemistry for the financial support. Technical support from MUN Genomic and Proteomics
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