# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 2 Materials and Methods
### Laboratory Experiment
#### 2.3.2 Protein Hydrolysis  
Protein hydrolysis was conducted according to the protocol described in detail by [PERSON] and [PERSON] [49]. Briefly, protein samples were dissolved in a 6 M urea solution in 0.1 M Tris-HCl buffer with pH = 7.8 to the concentration 10 mg/mL. Samples were centrifuged (2700\(\times\)\(g\), 30 min, 4 \({}^{\circ}\)C). Supernatants were reduced (5 uL of a 0.2 M dithietitol solution in Tris-HCl buffer per 100 uL of sample solution, 60 min, 57 \({}^{\circ}\)C) and alkylated (20 uL of 0.2 M iodoacetamide in Tris-HCl buffer per sample after reduction, 60 min, 21 \({}^{\circ}\)C).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample** & **Product Name** & **Kind of Processing** & **Content** \\ \hline \multirow{8}{*}{Sample 1.} & & & Indian Ocean squid (_Loligo duvaucelli_; kid78425) 1; \\  & & & Argentinian squid (_Illex argentiums_; kid6628) 1; \\  & & & Chilean mussel \\  & & & (_Mytilus chilensis_; toid173667) 1; \\  & & & Kadal shrimp (_Metapaneus dobsoni_; toid1378283) 1; \\  & & & Kiddi shrimp (_Parapaneus stylifera_; toid1288904) 1; \\  & & & Jinga shrimp (_Metapaneus affinis_; kid228858) 2 \\ \hline \multirow{2}{*}{Sample 2.} & & & Kiddi shrimp \\  & & & (_Parapaneus stylifera_; toid1288904) 1 \\ \cline{2-3}  & & & Whiteleg shrimp (_Litopenaeus vannamei_; kid6689) 3 \\ \hline \multirow{2}{*}{Sample 4.} & & & Chilean mussel \\  & & & (_Mytilus chilensis_; toid173667) 1 \\ \cline{2-3}  & & & \\ \hline \multirow{2}{*}{Sample 5.} & & & Kiddi shrimp \\  & & & (_Parapaneus stylifera_; toid1288904) 1 \\ \cline{2-3}  & & & Whiteleg shrimp (_Litopenaeus vannamei_; kid6689) 3 \\ \hline \multirow{2}{*}{Sample 6.} & & & Chilean mussel \\  & & & (_Mytilus chilensis_; toid173667) 1 \\ \cline{2-3}  & & & \\ \hline \multirow{2}{*}{Sample 7.} & & & \\ \cline{2-3}  & & & \\ \end{tabular}  
* Amino acid sequence of tropomyosin unavailable in the UniProt database (January 2024). 2 Fragments of sequence are available in the UniProt database (Access. No: A0A894 JSG1) (accessed 20 January 2024). Nine separate fragments containing up to 27 amino acid residues have been merged into one sequence with 151 residues. There are missed fragments up to c.a. 26 residues within sequence. 3 Entire amino acid sequence of tropomyosin available in the UniProt database (accessed 20 January 2024).  
\end{table}
Table 1: Products used for laboratory experiments.  
Solutions after reduction and alkylation were mixed with 775 \(\mu\)L of distilled water and 100 \(\mu\)L of a trypsin (Sigma-Aldrich, St Louis, MO, USA, cat. No T1426) solution (0.5 mg/mL, 10,000 BAEE units/mg of protein, where 1 unit produces \(\Delta\)A = 0.001 per minute at a wavelength of 253 nm with N\(\alpha\)-benzoyl-L-arginine ethyl ester as a substrate, at pH = 7.6 and a temperature of 25 \({}^{\circ}\)C in a reaction volume of 3.2 mL). Hydrolysis time was 17 h, and the temperature was 37 \({}^{\circ}\)C.  
Hydrolysates and unhydrolyzed proteins were purified according to [PERSON] et al. [50] using the solid-phase extraction (SPE) method by means of Supelclean LC-18 SPE Tube columns (Supelco, Bellefonte, PA, USA) with 3 mL volume of C18 stationary phase (particle size 0.45 \(\mu\)m, pore size 60 A). The washing sequence was as follows: conditioning using 6 mL of 0.1% trifluoroacetic acid (TFA) in water, equilibration using 6 mL 0.5% TFA, loading of the samples (acidified using TFA to final concentration 0.1%), washing using 0.5% TFA in water (3 times) and elution using 5 mL of acetonitrile. Samples were then dried in a vacuum concentrator ([PERSON], Osterode am Harz, Germany), dissolved in water, lyophilized and stored at \(-\)86 \({}^{\circ}\)C.  
Hydrolysis was monitored using SDS-PAGE electrophoresis according to Laemmli [51] protocol.