# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 1 Introduction  
Tropomyosins from seafoods (crustaceans and mollusks) belong to the key food allergens [1, 2, 3, 4], are considered causative agents of primary sensitization [5] and show cross-reactivity [6]. Crustacean and molluscan tropomyosins reveal also cross reactivity with their homologs from parasites [6, 7] and from edible insects [6, 8]. Apart from tropomyosins, some other proteins from seafoods also reveal allergenicity [6, 9, 10, 11].  
Amino acid sequences of invertebrate tropomyosins are strongly conserved. They possess, e.g., many common linear epitopes [12]. Sequence similarity or identity between linear epitopes from various allergens is considered to be one of the factors causing cross-reactivity [13].  
Traditional heat treatment (cooking or frying) is known to reduce protein allergenicity, although some allergens are thermostable [14]. Relatively high thermostability is an important feature of tropomyosins [15, 16, 17, 18, 19]. Heat processing, e.g., frying, can even increase the IgG/IgE-binding capacity of oyster, prawn and shrimp tropomyosins [18, 20, 21].  
Tandem mass spectrometry has recently been deemed a standard method for the identification of peptides and proteins including allergens [22; 23]. It is widely used, e.g., for tropomyosin identification and quantitation [19; 24; 25; 26; 27; 28].  
[PERSON] and coworkers [19] carried out peptidomic analysis of allergens of shrimps (_Litopenaeus vannamei_) subjected to baking or frying. The number of peptides being products of tryptic digestion of tropomyosin in extracts from boiled and fried shrimps was much smaller than in extracts from raw shrimps. The same finding concerned other allergens. This phenomenon may be explained by chemical modifications of proteins during heat treatment, e.g., making lysine or arginine residues inaccessible for trypsin.  
According to standard recommendations, the presence of numerous common fragments in tropomyosin sequences makes it difficult to choose peptide markers specific for particular species [29]. On the other hand, it enables the identification of peptides from proteins belonging to the tropomyosin family, even if the entire protein sequence is unknown, according to the strategy named comparative proteomics [30; 31]. This strategy attributes a peptide marker not to a single protein but to a group of homologs belonging to the same family. Examples of protein markers attributed to more than one allergenic protein have been presented in a review article published by [PERSON] et al. [32]. Markers occurring in sequences of proteins from various species have been applied to detect carp and herring proteins [33], as well as mammalian and bird meat proteins [34].  
There are two major proteomic strategies recommended for the detection of allergens in foods: targeted and untargeted proteomics [35]. The first one involves the prior selection of peptides being markers of the occurrence of allergenic proteins and is recommended for the detection of known allergens. The second one involves the identification of peptides by mass spectrometry followed by database screening for proteins containing the sequence identified or the fragment as similar as possible and is recommended for the detection of new allergens. In the case of strongly conserved protein families, e.g., tropomyosins, we can posit another viable means: application of selected peptides to detect allergens with both known and unknown sequences.  
The aim of the present study was to evaluate the possibility of identifying allergenic tropomyosins with known and unknown amino acid sequences in seafoods, including the influence of boiling and frying processes.