# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 3 Results  
Distribution of the peptides chosen as markers of allergenic tropomyosins among proteins belonging to this family (defined according to IntePro database) is presented in Figure 2. There was one peptide among the 76 compounds resulting from in silico proteolysis: AADESER occurring in over 30% of tropomyosins, five peptides found in 5-30% of tropomyosins and four found in 1-5% of tropomyosins. To compare: 21.6% of tropomyosins contain at least one known epitope common to more than one sequence of protein belonging to this family [12]. Tropomyosin sequences show a high degree of similarity among various crustacean species (ranging between 88 and 100%) and among various mollusk species (ranging from 70 to 97%), and lower between crustaceans and mollusks (between 56 and 65%) [1; 9]. For this reason, it is more difficult to find peptides, which are products of trypsin hydrolysis, that will be common to the tropomyosins of mollusks and crustaceans than for each of these  
Figure 2: Percentage of proteins belonging to the tropomyosin family (ID: IPR000533 in the InterPro database), containing predicted products of simulated hydrolysis by trypsin. Peptides present in at least two tropomyosins are included. Data visualized in this figure are shown also in Table S2 in the Supplementary Materials. groups separately. The in silico analysis showed that it was possible to isolate peptides that are characteristic of crustaceans, mollusks and those that occur in both groups. Among the identified peptides, 61 contained from 7 to 10 amino acid residues, 10 between 11 and 15 amino acid residues and 5 above 15. The maximum length of the selected peptides was 21 amino acid residues. The length of a peptide marker recommended in a previous study [33] was 7-15 amino acid residues. An example of the MS/MS spectrum of an identified peptide (with the MDALEN-QLK sequence) is provided in Figure 3. Chromatograms of protein extracts and their hydrolysates are included in the Supplementary Materials in Figures S7-S20. Mass spectra contain typical ions resulting from the fragmentation of peptide bonds and products of water or ammonia release from them (neutral loss) [57; 58]. Modifications of amino acid residues in individual peptides (oxidation, deamidation, etc. are summarized in Table S3 in the Supplementary Materials). The length of peptides varied from 7 to 41 amino acid residues. Spectra of the two longest peptides detected during the experiment (34 and 41 amino acid residues) are shown in Figures S7 and S8 in the Supplementary Materials. Contemporary proteomic and peptidomic studies involve the detection of long peptides (with length exceeding 20-25 amino acid residues) [59; 60; 61]. Distribution of peptides among the samples is shown in Figure 4. Peptides detected in hydrolysates prepared from particular samples are divided into three categories: predicted peptides, longer peptides containing predicted sequences as fragments and unpredicted peptides. The boiling of seafood samples led to an increase in peptide length as compared with the unprocessed samples. Tandem mass spectrometry is considered as a method for the identification of an allergen sequence but proves inefficient in detecting denaturation if there is no chemical modifications [14]. On the other hand, changes in the secondary and tertiary structure may affect susceptibility of proteins to enzymatic hydrolysis, resulting in missed cleavages and hence an increase in the length of peptides in protein hydrolysates [63]. Peptides identified in the boiled samples contained a higher number of entire linear epitope sequences than those found in the raw samples. In contrast, such epitopes were absent in the peptides identified in the fried samples (see Figure 4). Figure 3: Example of the MS/MS spectrum of a tropomyosin marker, with the MDALENQLK sequence, generated by the PEAKS program. Nomenclature of fragment ions according to [PERSON] and [PERSON] [62].