# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 4 Discussion  
Proteomic methods based on peptide markers offer a promising alternative to routine methods for assessing food authenticity, such as DNA analysis or immunological tests [74]. The rapid development of databases makes it possible to identify a significant number of proteins with unknown sequences (such as these summarized in the Table 1) based on already described sequences, especially when they belong to highly conserved protein families [31]. On the other hand, it is increasingly difficult to apply the traditional protein identification strategy based on searching for peptides unique to a given protein sequence. This strategy is recommended when identifying allergens [29]. Searching for markers based on the above recommendation requires knowledge of the sequence of a given protein and the selection of peptides unique to a given amino acid sequence. Scientific publications mention its \"specificity\" as one of the features of a peptide marker [29]. The existence of homologous proteins with high sequence similarity (including the unknown ones) hampers meeting the above recommendations [75]. In the case of highly conserved protein families (with high amino acid sequence similarity), the same peptide may occur in proteins of different species of living organisms. An example would be cow's milk protein markers, which also appear in the sequences of buffalo milk proteins (_Bubalus__bubalis_) [32], or peptides that are markers of myosins in herring (_Clupea__harengus_) and carp (_Cyprius__carpio_) [33]. The second myosin sequence was not annotated in the UniProt database. Currently, the use  
\begin{table} \begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Category of Peptides 1**} & \multicolumn{5}{c}{**Percentage of Proteins of Given Tax (mean \(\pm\) SB 2**} \\ \cline{2-6}  & _Crustacea_ & \begin{tabular}{c} **Other** \\ _Arlrhopa_ \\ \end{tabular} & _Mollusca_ & \
\begin{tabular}{c} **Other** \\ _Metaza_ \\ \end{tabular} &
\begin{tabular}{c} **Other** \\ _Metaza_ \\ \end{tabular} \\ \hline Peptides characteristic for _Crustacean_ (n = 15) & 95.3 \(\pm\) 0.8 & 3.2 \(\pm\) 0.7 b  & 1.2 \(\pm\) 0.2 b  & 0.1 \(\pm\) 0.1 a  & 0.2 \(\pm\) 0.2 a  \\ Peptides characteristic for _Arlrhropoda_ (n = 19) & 6.3 \(\pm\) 1.0 a  & 8.7 \(\pm\) 2.6 & 1.4 \(\pm\) 0.7 b  & 8.5 \(\pm\) 2.1 a  & 0.18 \(\pm\) 0.04 b  \\ Peptides characteristic for _Mollusca_ (n = 28) & 0.06 \(\pm\) 0.03 a  & 0.2 \(\pm\) 0.1 a  & 95.2 \(\pm\) 1.4 & 3.4 \(\pm\) 1.2 & 1.1 \(\pm\) 0.5 b  \\ Inconclusive peptides (n = 14) & 0.1 \(\pm\) 0.1 a  & 3.6 \(\pm\) 2.2 a  & 0.7 \(\pm\) 0.5 a  & 28.4 \(\pm\) 9.6 & 67.3 \(\pm\) 9.1 \\ \hline \hline \end{tabular}   
* 1
* \(\mathrm{n}\)—number of peptides present in at least 10 proteins. 2
* \(\mathrm{SE}\)—standard error. a of peptides occurring in more than one sequence as markers is considered acceptable in the case of allergens with highly similar amino acid sequences [25; 76; 77]. Invertebrate tropomyosins are an example of a family of proteins containing numerous common fragments, including epitopes [12]. The research by [PERSON] et al. [26], [PERSON] et al. [76] and [PERSON] et al. [77] focused on finding peptides that could be used to detect crustacean tropomyosins, eliminating the risk of obtaining a false-positive result (peptides not present in the tropomyosin sequences of mollusks and insects). In the aforementioned publications, such peptides are called \"signature\" or \"specific\" and are present in more than one crustacean tropomyosin sequence. If raw material of a food product is known (e.g., a specific species of shrimp), an approach using peptides specific to the shrimp protein sequence is justified. However, if the product contains a shrimp (or other invertebrate) of an unknown species or with an unknown tropomyosin sequence that does not contain the peptide selected as \"specific\", an attempt to detect the presence of the protein in the product may result in a false negative result.