Peptidomics analysis of simulated mouth, gastric and intestinal digestion pattern of allergenic tropomyosin

The digestion patterns of allergenic proteins play a key role in allergenicity. In order to completely remove allergenic properties of a protein it is necessary a complete enzymatic digestion. Whether the enzymatic digestion is not efficient, the persistence of bigger peptides can occur and put the basis for the development of the sensitization process. The hypothesis of the current work is based on the probability that shrimp tropomyosin (TM) is not fully digested or presents a digestion pattern that generates some peptides that can be immunogenic. Therefore, the work plan designed aims to study the cleavage pattern of respectively purified chicken TM, recombinant chicken TM, purified TM of Penaeus monodon (Pen m 1), recombinant TM of Penaeus monodon (rPen m 1) and recombinant TM of Crangon crangon (rCrac c 1). One mg of each orthologue has been processed through simulated mouth, gastric and intestinal digestion. The sample was frozen after this step, concentrated and cleaned through protein precipitation. Afterwards, the protein pellet was analyzed for peptidomic analysis through 1D tricine gel electrophoresis, 2D tricine gel electrophoresis and mass spectrometry. Shrimp TM digestion pattern highlighted the presence of a resistant band at an average MW of 25 kDa that could be involved in the immunogenic process. This approach (peptidomics through 1D-2D Tricine/MS) could represent a milestone for the study of digestion patterns of allergenic proteins or for the study of allergenic potential of novel foods. Authors are grateful to COST Action FA1402 entitled: Improving Allergy Risk Assessment Strategy for New Food Proteins (ImpARAS)