# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 4 Discussion
The Unipept program was used post experiment to determine the distribution of the identified peptides among protein families and taxonomic lineages of organisms producing these proteins. In turn, BLAST [40] and Unipept [43] programs were used to determine the distribution of peptides among proteins, protein families and taxa. Based on study results, the second one may be recommended as it provides a summary of information about any peptide (list of proteins with links to the UniProt database, list of all InterPro families) and enables displaying data as a table or as a lineage tree. The Unipept is thus more user-friendly than BLAST. Planning and interpretation of sequence analysis via bioinformatics tools should take into account a problem defined as the continuity of operation [93]. Protocols of bioinformatics analysis should be adapted to the changes in available tools. Options for UniProt database screening using tools such as PSI-BLAST [40], applied in this study (see Section 2.1.3) and in our previous studies [12; 32; 41], are no longer available. Recently, sequence analysis tools are accessible at the European Bioinformatics Institute via the EMBL-EBI Job Dispatcher service ([[https://www.ebi.ac.uk/jdispatcher](https://www.ebi.ac.uk/jdispatcher)]([https://www.ebi.ac.uk/jdispatcher](https://www.ebi.ac.uk/jdispatcher)); accessed 30 September 2024) [94]. Database screening using short peptide sequences as queries may be performed using the PAM70 matrix instead of the PAM30 one. The \"Expected threshold\" parameter is no longer available. The parameters adjustable by user include \"E-cutoff\" and \"Results E limit\". The E value is interpreted as the probability that that sequence alignment is incidental. E values, obtained during aligning short sequences, are extremely high as compared with these obtained for protein sequence alignments (>100 amino acid residues). We can thus recommend maximum values of these parameters. In the case of the search for short sequences, identical with query (100% identity and the same length) E value is not applicable as the criterion of alignment quality. PAM30 matrix and expected threshold as adjustable parameter is still available for NCBI-BLAST program at the same website. Generally, default parameters of tools available via the EBI-EMBL job dispatcher are optimized for screening database using protein sequences as queries. The results presented here may constitute the starting point to discuss current recommendations for choice of peptide being possible markers for identification of allergenic proteins using mass spectrometry, supported by bioinformatics. The above recommendations have been recently summarized in the review article of [PERSON] and coworkers [95]. The first recommendation assumes that marker peptides should be specific and unique to allergenic protein. This goal is not achievable in the case of invertebrate tropomyosins if we look for peptides, being the fragment of only one protein sequence, annotated in the UniProt database. Peptides may be unique for protein family defined according to the InterPro database (in this experiment, tropomyosin family-ID: IPR000533). Individual peptides are not assigned to multiple species for instance classified according to NCBI Taxonomy database) but to broader taxonomic units such as _Crustacea_ (subphylum), _Arthropoda_ (phylum) or _Mollusca_ (phylum). Unipept program is a sufficient tool to determine taxonomic lineage of individual peptide. Peptides with defined taxonomic lineages (attributed to defined taxon) may be recommended as markers of allergens. They can be found in proteins of unknown or incomplete sequences (samples 1, 2 and 4 in Table 1). Peptidespossessing inconclusive (undefined) taxonomic lineages are not useful in the context of protein identification. The second recommendation concerns marker peptide length. The preferred range is between 7 and 20 amino acid residues.