## 3 Results and Discussion
### Effects of OM Treatment on Protein Oxidation
#### 3.3.2 Oxidation Sites on Proteins  
When food proteins are exposed to inactive molecular oxygen and ROS such as ozone, their interaction leads to oxidative modification situated at aliphatic, aromatic, heteroatom, or backbone sites [41; 42]. To compare the degree of oxidation in proteins of the two treatments, HR-MS was used to detect the possible oxidative products and their exact positions. * Types of oxidative modifications B and y ion analysis is performed to detect protein oxidation sites and oxidation modification types. An example can be seen in Figure 2, where the MS/MS spectrum of the peptide KVLGNPSNEEM(O)NAK derived from MLC1f protein of pork samples and the peptide GPP(O)GPMGPPGLAGPPGEPGR from collagen type I alpha 1 protein of fish samples are shown, indicating that the same oxidative modification occurs in the CW and OM groups. Specifically, peptides of pork protein with Met monooxidation were found in the two treatments (Figure 2A,B), while peptides of fish protein with Pro monooxidation were found in the two treatments (Figure 2C,D). Figure 2: Representative examples of oxidatively modified peptides which were identified. MS/MS spectrum of the peptide KVLGNPSNEEM(O)NAK derived from pork samples treated with CW (**A**) and OM (**B**); MS/MS spectrum of the peptide GPP(O)GPMGPPGLAGPPGEPGR derived from fish samples treated with CW (**C**) and OM (**D**). CW: conventional water-washing; OM: ozone-microbubbles-washing. As shown in Figure 3A, the same eight oxidative modification types were detected in pork and fish samples with the CW and OM treatments. The numbers of oxidatively modified peptides detected in pork with CW and OM treatment are 232 and 277 (Tables S1 and S2), respectively. The numbers of oxidatively modified peptides detected in fish samples with CW and OM treatment are 505 and 489 (Tables S3 and S4), respectively. It shows that OM treatment has a slightly greater impact on the oxidation of pork protein, and the effects of OM treatment on the oxidation of fish protein is the same as that of CW treatment. In addition, the oxidative modifications of monooxidation, dioxidation, and trioxidation ranked as the top three oxidative modifications in both pork and fish samples with the two treatments. The modifications of Histidine oxidation to Aspartic Acid (Asp) and Asparagine (Asn) and oxidation to nitro also frequently occurred in various samples. For pork samples (Figure 3B, Tables S1 and S2), the former three types of oxidative modification, as a whole, account for 93% and 87% of the entire modified peptides in the CW and OM groups, respectively. Compared with the two treatments, CW was more likely to promote the monooxidation of Met, Asp, Tyr, and Trp; OM was more likely to promote the monooxidation of Pro, Asn, His, Lys, Arg, and Cys, and the number of dioxidation and trioxidation in the OM group was slightly higher than that in the CW group on each modification site. In both CW and OM groups, most sites underwent monooxidation rather than dioxidation and trioxidation. Met, Pro, and Asp were the primary amino acid residues most prone to oxidation. Moreover, higher oxidation levels such as dioxidation and trioxidation occurred mainly in Phe and Tyr. This result was consistent with the previous study [43], which reported that dioxidation was prone to occur in aromatic amino acids. The regularity of the oxidative modification sites was also found in fish samples (Figure 3C, Tables S3 and S4), although the number of modifications of individual amino acids residues such as Pro varied. * Types and properties of oxidatively modified proteins of pork samples  
A total of 51 and 56 species of proteins were oxidized in the CW and OM groups, respectively. The detected peptides with oxidative modification were mainly derived from myosin, actin, and collagen in the two treatments (Figure 4A). The largest number of peptides with oxidative modification were derived from different myosin family members. Myosin was prone to oxidize compared with actin, which was mainly due to the different structures [44].