# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 4 Discussion
Part of unpredicted peptides, present especially in the fried samples, may be attributed to proteins from diverse, unrelated families and diverse taxa. This finding concerns especially peptides with limited length (Figure 6) and is consistent with findings from our previous research [32; 41] into the distribution of hexa- and heptapeptides among proteins and their families. Unpredicted peptides may be attributed to animals other than arthropods and mollusks (e.g., nematodes or vertebrates) or to bacteria. The latter could be found during proteomic analysis of larval gut proteome of insects [65] or in fermented foods [88] but are unlikely in fried samples. Taxonomic lineages attributed to taxa that are unlikely to be present in the samples are considered here as inconclusive. It should be emphasized that the results obtained for the fried samples did not confirm those obtained for the raw or boiled samples. Peptides with inconclusive lineages, found in the fried samples, were often recognized as chemically modified (deamidated or oxidized). Based on the above results, the workflow including identification of protein fragments should not be recommended for the analysis of fried food samples. We can point out an advantage of the analysis of peptides from boiled samples. Allergenic proteins can also be detected, based on epitope sequences. One of the criteria for recognizing a protein as an allergen is the presence of at least two IgE-binding epitopes in its sequence [89]. The identification of at least two peptides containing entire epitope sequences suggests that the product contains proteins fulfilling the above criterion. The above criterion is much less restrictive than those recognized by the International Union of Immunological Societies (IUIS) [90], according to which, an allergenic protein should meet multiple biochemical and immunological criteria. The allergen should have a known sequence and known post-translational modifications (if any) and should be purified to be homogeneous or nearly homogeneous. Characterization of an allergenic protein should include the determination of its basic physicochemical properties, such as molecular weight and isoelectric point, as well as obtaining monoclonal or monospecific antibodies interacting with the compound being the object of research. Immunological criteria for allergenicity include the determination of the frequency of serum IgE antibodies in as many patients as possible (at least 50 are recommended), the determination of allergenic potential (e.g., skin tests), reduction in the binding capacity of IgE antibodies after removal of the allergen and the IgE-binding capacity of the recombinant allergen. The standard allergen analysis strategy assumes, among others, the use of protein standards that meet the above criteria [76; 91]. Detection of epitopes from allergenic proteins cannot meet the most restrictive criteria but may be sufficient for a respective health claim. Identification of epitopes is much more likely in boiled than in unprocessed samples (see Figure 4). The search for epitopes occurring in proteins from few or several species is considered as an emerging trend in immunology [92]. In turn, the identification of protein fragmentscontaining epitopes may be deemed a preliminary step before implementing protocols involving more restrictive criteria of allergenicity [80]. To summarize, two bioinformatics strategies were applied in the study: selection of potential markers before experiment and determination of distribution of detected peptides among protein families and taxonomic lineages. A priori selection was successful in the case of unheated products and failed in the case of the fried ones (Figure 4). In the boiled products, the predicted peptides were often fragments of the longer ones (Figure 4). Boiling leads to the appearance of missed cleavages, i.e., bonds resistant to proteolysis, although they are predicted to be susceptible to this transformation.