Characterization of airborne allergens of selected crustaceans and monitoring of their occupational exposure levels using mass spectrometry

Abdel Rahman, Anas M. (Anas Mahmoud) (2011) Characterization of airborne allergens of selected crustaceans and monitoring of their occupational exposure levels using mass spectrometry. PhD thesis, Memorial University of Newfoundland.

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Abstract

The number of workers in the processing and harvesting sector of the fishery has increased substantially in recent decades, seeing more than 41 million workers worldwide engaged in various activities related to seafood production. Serious allergies and asthma have developed in the seafood occupational workplace as the result of airborne allergenic proteins being aerosolized on fishing vessels and in processing plants. Traditionally, the identification and quantification of these allergens are performed by immunoactive-based techniques such as radioallergosorbent test and enzyme-linked immunosorbent assay. -- In this study, a novel strategy of monitoring airborne seafood allergens was developed using tandem mass spectrometry. First, the major allergenic proteins of snow crab and black tiger prawn were identified and profiled using an allergeomics approach. The immunoblotting was performed against sensitized patients' sera and the active protein bands were characterized using peptide mass fingerprinting mass spectrometry. The purified forms of the target allergenic proteins, tropomyosin and arginine kinase, were used as reference materials for primary structural identification and for quantification method development. -- For protein sequencing, the samples were exposed to different enzymatic digestions (Trypsin, Glu-C V8, and Asp-N), the peptides analyzed by different mass spectrometry ion sources, and various derivatization protocols were used to maximize the amino acid sequence coverage. The product mass spectra of the enzymatically produced peptides were uploaded to a Mascot search engine and the national center for biotechnology information non-redundant database searched. Signature peptides that surrogate each individual allergenic protein were determined and chemically synthesized (purchased from third party) for use as analytical standards in both light and heavy forms. Subsequently, a quantification approach using liquid chromatography-multiple reaction monitoring isotopic dilution tandem mass spectrometry was developed to measure snow crab tropomyosin and arginine kinase in air samples. A comprehensive validation study was performed for the quantification method with respect to the international conference on harmonization guidelines. The air sampling protocol, allergen extraction recovery, and the completeness of the protein digestion were optimized for real air sample collection. Actual air samples were collected from a simulated crab processing plant and analyzed by mass spectrometry.

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