## 3 Results and Discussion
### Effects of OM Treatment on Protein Oxidation
#### 3.3.2 Oxidation Sites on Proteins
Take myosin-1 as an example. With the highest proportion of oxidative modification, the main oxidative types were monooxidation and dioxidation in both treatments. In addition, modifications of Histidine oxidation to Asparatic Acid and Asparagine and oxidation to nitro were found in the CW group (Figure 4B). The numbers of total oxidative modifications in peptides of the CW group were similar but slightly higher than that of the OM group. In both CW and OM treatment groups, Met was the most vulnerable amino acid residue to oxidation because of the high activity of  
Figure 3: The number of oxidative modifications among all samples (**A**); oxidation sites and the number of monooxidation, dioxidation, and trioxidation modifications in pork sample (**B**); and fish sample (**C**). CW: conventional water-washing; OM: ozone-microbubbles-washing. M: Methionine; D: Aspartic Acid; P: Proline; N: Asparagine; Y: Tyrosine; K: Lysine; C: Cysteine; H: Histidine; W: Tryptophan; R: Arginine; F: Phenylalanine. sulfur atoms [43]. In addition, Pro was more prone to dioxidation in the OM group, and monooxidation in the CW group. As is listed in Tables S1 and S2, for some sequence of peptides, such as EDQVFPMNPPKFDK, monooxidation occurred mainly in the CW group (EDQVFP(+15.99)MN(+15.99)PPKFDK, EDQVFP(+15.99)M(+15.99)NPPK), while dioxidation occurred in the OM group (EDQVFP(+31.99) MNPPKFDK). Since different types of oxidations occurred in similar sequences, the results of oxidative modification in both proteins were similar. From this aspect, OM treatment did not cause more severe oxidation. For actin, take actin, alpha skeletal muscle as an example. Six types of oxidative modification were detected in both CW and OM groups (Figure 4C). Monooxidation, deoxidation, and trioxidation were the main types of oxidative modification, although the sensitivity of different amino acids residues varied in the CW vs. OM comparison. In addition, Met was the dominant site in CW and OM groups with the modification of monooxidation and dioxidation [45]. * Types and properties of oxidatively modified proteins of fish samples As for the fish sample, 61 and 64 species of proteins were oxidized in CW and OM groups, respectively, and the detected peptides with modification were mainly derived from myosin and collagen (Figure 5A). Figure 4: The number of oxidative peptides and the protein which they derived from (**A**); the type and the number of oxidative modifications in myosin-1 (**B**); the type and the number of oxidative modifications in actin, alpha skeletal muscle (**C**); CW: conventional water-washing; OM: ozone-microbubbles-washing. M: Methionine; D: Asparatic acid; P: Proline; N: Asparagine; Y: Tyrosine; K: Lysine; C: Cysteine; H: Histidine; W: Tryptophan; F: Phenylalanine. The protein name was according to the Uniprot database. As an essential structural protein in fish, mainly distributed in fish skin and bone [46], collagen contains more modifications than its counterpart proteins. Take collagen type I alpha 2 as an example. Both monooxidation and dioxidation were detected in the two treatments. The main modification sites in both treatment groups were Pro and Lys with monooxidation (Figure 5B). For these amino acid radicals, the mass shift of oxidation and hydroxylation was similar. Since hydroxylation was the critical type of modification in collagen, which occurred mainly at the Pro and Lys sites [47], it suggested that the main modification of Pro and Lys here was hydroxylation, which benefited collagen stability. In addition, the number of oxidative modifications in amino acid residues detected was roughly the same in the CW vs. OM comparison. For myosin, take myosin heavy chain, fast skeletal muscle as an example. Six types of oxidative modification were detected in both CW and OM groups, where the monooxidation of Met and Asp was prone to occur (Figure 5C). The oxidative modification of myosin of fish samples was similar to that of pork samples.