# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 4 Discussion
In such a case, it is better to use a peptide that is present in as many crustacean (or other invertebrate) tropomyosin sequences as possible. This increases the probability of detecting shrimp proteins in the food product. This approach, proposed in this work, stands in opposition to the traditional allergen detection strategy, which involves searching for peptides unique to a given protein amino acid sequence. If the selected marker is universal for the tropomyosin sequences of many seafood species, it also allows for the detection of proteins in the food product that have not been described in the literature and whose sequence is unknown. Moreover, if the marker is universal for many proteins from the tropomyosin family (not only from mollusks or crustaceans), it can be used to detect potential allergens in food containing, for example, insect proteins. A useful feature of such a peptide marker is its association with the amino acid sequence of the tropomyosin epitope. Peptide markers whose amino acid sequences are also fragments of epitope sequences or entire epitopes may be a useful tool for detecting allergenic proteins. The IEDB database contains fewer epitopes from _Mollusca_ than from _Crustacea_. Therefore, in the case of mollusk tropomyosins, finding a peptide marker that is also a fragment or an entire epitope is more difficult than in the case of crustacean tropomyosins. The data presented here show that the entire sets of peptides characteristic for _Crustacea_ and _Mollusca_ are highly specific for these taxa. The percentage of proteins derived from organism belonging to these taxa is ca. 95% (see Table 2). This percentage can be interpreted as 95% probability of true positive result (and 5% probability of a false-positive result) if we are detecting proteins from _Crustacea_ or _Mollusca_ in our products. The category of peptides characteristic for _Arthropoda_ covers a broader range of organisms but is less specific than the previous two categories. Only ca. 90% of proteins containing detected peptides as fragments were detected in _Arthropoda_ samples (Table 2). About 8.5% of proteins originated from other _Metazoa_. Such a result may be interpreted as the probability of false-positive result. Peptides of inconclusive taxonomic lineages may be attributed with high probability to organisms other than Arthropoda and Mollusca. The allergen identification strategy based on searching for peptide markers common to a larger number of proteins can be applied to identify not only tropomyosins but also other highly conserved families, e.g., allergens derived from milk or eggs [32]. This strategy is, however, unsuitable for protein families with highly variable sequences, such as parvalbumins, which have been recommended as precursors of markers specific to particular fish species [78; 79]. The present study results indicate that the presence of common fragments containing epitopes in tropomyosins may be helpful in identifying allergens from crustaceans and mollusks in seafoods via other methods, such as immunochemical assays or PCR. Suchmethods have been recently developed and deployed in several experiments [80; 81; 82; 83; 84; 85; 86] and are also viable for peptides resulting from the hydrolysis of the boiled products and containing entire epitopes within their sequences. Taking into account the above results, the boiling of the samples may be recommended as part of the protocol of sample preparation before MS/MS analysis. The use of fried samples cannot be recommended in contrast to the unheated and boiled ones, due to the fact that mainly peptides with inconclusive taxonomic lineage were detected during hydrolysis of proteins from seafood after frying. Thus, it may be concluded that frying seafoods poses serious difficulties in the detection of allergenic tropomyosins, based on the identification of peptides being their fragments. However, air-frying applied in the present experiment is considered as less destructive to food components (e.g., proteins) than other frying methods [87].