## 2 Results and Discussion
### Preparation of Protein Hydrolysate from Hairtail (T. japonicus) Muscle (HTP)
#### 2.2.3 Gel Filtration Chromatography (GFC) of DE-3  
GFC is a well-accepted separated technique on the basis of molecular size and usually applied to either fractionate molecules and complexes in a sample into fractions with a particular size range, or remove salt from a preparation of macromolecules [3; 36]. Therefore, GFC is often used to separate peptides from protein hydrolyses and their fractions [6; 7].  
As shown in Figure 5A, DE-3 was separated into two fractions of DE-3-1 and DE-3-2 using a Sephadex G-15 column, and each fraction was collected, lyophilized, and then evaluated for DPPH\(\bullet\) and HO\(\bullet\) scavenging activity. Figure 5B indicated that DPPH\(\bullet\) and HO\(\bullet\) scavenging activities of DE-3-2 were 69.21 \(\pm\) 0.91% and 84.16 \(\pm\) 1.26% at the concentration of 5.0 mg protein/mL, which were significantly higher than those of DE-3 (DPPH\(\bullet\) 58.91 \(\pm\) 1.89%; HO\(\bullet\) 69.74 \(\pm\) 2.61%) and DE-3-1 (DPPH\(\bullet\) 36.27 \(\pm\) 0.79%; HO\(\bullet\) 41.38 \(\pm\) 1.49%) (\(p\) < 0.05). Therefore, fraction DE-3-2 was selected for the following isolation process.  
Figure 4: Elution profile of HTP-I in DEAE-52 cellulose anion-exchange chromatography (**A**) and radical scavenging activity of HTP-I and its four subfractions at the concentration of 5.0 mg protein/mL (**B**). All data are presented as the mean \(\pm\) SD of triplicate results. \({}^{a-f}\) or \({}^{A-E}\) Values with same superscripts of this type indicate no significant difference (\(p\) > 0.05).  
2.4 Isolation of Peptides from DE-3-2 by Reverse-Phase High Performance Liquid Chromatography (RP-HPLC)  
RP-HPLC is an effective technique applied to purify and quantify peptides in a mixture solution on their hydrophobic character [37]. The retention time (RT) can qualitatively analyze the isolated peptide and adjusted by changing the ratio of methanol or acetonitrile in mobile phase, and the peak area can be used for quantitative analysis of the isolated peptide [3; 38]. As shown in Figure 6, DE-3-2 was finally purified using RP-HPLC system on an Agilent 1260 HPLC system with a Zorbax C-18 column, and the eluted peptides were gathered separately in accordance with chromatographic peaks. Among all chromatographic fractions, eight peptides with RT of 8.919 min (TJP1), 9.189 min (TJP2), 12.112 min (TJP3), 13.829 min (TJP4), 14.209 min (TJP5), 17.237 min (TJP6), 19.772 min (TJP7), and 20.436 min (TJP8) showed high radical scavenging activities. Therefore, TJP1-TJP8 were collected and lyophilized for amino acid sequence identification and activity evaluation.  
Amino Acid Sequence Analysis and Mass Spectrometry of Peptides from Protein Hydrolysates of Hairtail Muscle  
For more detailed discussion on the structure-function relationship, the amino acid composition, sequences, and molecular mass of eight isolated peptides (TJP1-TJP8) were determined using protein sequencer and ESI-MS, and the results were shown in Figure 7 and Table 1. The amino acid sequences  
Figure 5: Elution profile of DE-3 in Sephadex G-15 chromatography (**A**) and radical scavenging activities of DE-3 and its fractions at 5.0 mg protein/mL concentration (**B**). All data are presented as the mean \(\pm\) SD of triplicate results. \({}^{a-c}\) or \({}^{A-C}\) Column wise values with same superscripts of this type indicate no significant difference (\(p>0.05\)).  
Figure 6: Elution profile of DE-3-2 separated by RP-HPLC system on a Zorbax, SB C-18 column (4.6 \(\times\) 250 mm) from 0 to 40 min.  
of eight isolated peptides (TJP1-TJP8) were identified as Gln-Asn-Asp-Glu-Arg (TJP1), Lys-Ser (TJP2), Lys-Ala (TJP3), Ala-Lys-Gly (TJP4), Thr-Lys-Ala (TJP5), Val-Lys (TJP6), Met-Lys (TJP7), and Ile-Tyr-Gly (TJP8) with molecular weights of 660.3, 233.0, 217.1, 274.1, 318.0, 245.1, 277.0, and 351.0 Da, respectively, which were agreed well with their theoretical masses of 660.6, 233.3, 217.3, 274.3, 318.4, 245.3, 277.4, and 351.4 Da.