# Identification of Allergenic Tropomyosins with Known and Unknown Sequences in Seafood Products
## 2 Materials and Methods
### Laboratory Experiment
#### 2.3.3 Liquid Chromatography-Tandem Mass Spectrometry  
LC-MS/MS protocol was adapted from this presented by [PERSON] et al. [52]. The analysis was performed using an LCMS-9030 quadrupole time-of-flight (Q-TOF) mass spectrometer (Shimadzu, Tokyo, Japan) with ESI (electrospray) ionization. The assembly consisted of two LC-30 AD pumps, a DGU-20A5R degasser, a SIL30 ACMP autosampler, a CTO-20 AC thermostat, a CBM-20A controller, an NGM-11 nitrogen generator, and an SF2 FF compressor. Mass spectrometer was calibrated using Calibration Solution LCMS-9030, provided by Shimadzu (Part Number: 220-91239-91). A Jupiter Proteo(r) column from Phenomenex (Torrance, CA, USA) was used (150 mm \(\times\) 1 mm with C18 stationary phase, particle size of 5 \(\upmu\)m, pore size of 110 A). Solvent A--0.1% formic acid (LiChropur(r) Cat. No: 5.33002) in water (LiChroslov(r) Cat. No: 1.15333) (\(v/v\)) and solvent B--0.1% formic acid in acetonitrile (LiChroslov(r) Cat No 1.00029) (\(v/v\)) were used as mobile phases. Reagents of mass spectrometry grade were used. Gradient was as follows: start--5% B, 15 min--10% B, 20 min--30% B, 25 min--100% B, 26 min--100% B and 30 min--5% B. Data acquisition time was 30 min, flow rate was 0.05 mL/min, injection volume was 15 \(\upmu\)L and column temperature was 40 \({}^{\circ}\)C.  
The operating parameters of the mass spectrometer were as follows: heating gas flow rate: 10 L/min, nebulizing gas flow rate: 3 L/min, internal temperature of the apparatus: 300 \({}^{\circ}\)C, desolvation line temperature: 250 \({}^{\circ}\)C and drying gas flow rate: 10 L/min. The following method parameters were applied: positive polarization, needle voltage: 4 kV, loop time: 0.7 s, collision gas pressure: 230 kPa, accelerating voltage in the collision chamber: 35 \(\pm\) 10 V and ion accumulation mode: switched on. MS spectra were recorded in the m/z range of 300 to 1500 Da and spectra after fragmentation of precursor ions in the m/z range of 50 to 1500. The data-dependent acquisition mode (DDA) was used to select peptide precursor ions for fragmentation, which included four independent MS/MS fragmentation scans of selected precursor ions, preceded by a full MS scan. Precursor ions with an intensity of at least 300 counts were subjected to automatic fragmentation. The retention time of the peptides was determined after smoothing the chromatogram according to the algorithm described by [PERSON] and [PERSON] [53]. The obtained results were processed in LabSolutions version 5 software (Shimadzu, Tokyo, Japan). RP-LC-MS/MS analyses were performed in triplicate.  
Peptide identification was performed using PEAKS software [54] version 10.0 (Bioinformatics Solutions Inc., Waterloo, ON, Canada). A set of 13,298 tropomyosin sequences, downloaded from UniProtKB (on 9 July 2020), was used as a database. To reduce the probability of obtaining false-positive results, the parameters recommended by [PERSON] et al. [26] were used: \(-\)10 logP \(\geq\) 20 for peptides from the thawed and cooked products and \(-\)10 logP \(\geq\) 15 for peptides from the fried products (due to numerous amino acid modifications). Cysteine carbamidomethylation was set as constant modification, whereas methionine oxidation, as well as deamidation of glutamine and asparagine, were set as variable modifications [25; 55; 56].