*3.3. Physicochemical Properties Analysis* 3.3.1. Solubility

Solubility is one of the critical characteristics of food proteins, and is often used to represent the degree of cross-linking and aggregation to a certain extent. As shown in Table 2, with the increasing AAPH concentration, the solubility of WPI shows a significant downward trend (*p* < 0.05), and the lowest solubility (20.44%) was observed at 10 mmol/L AAPH. This may be due to the unfolding and destruction of WPI conformation by peroxy radical oxidation, then further polymerization occurs to form macromolecule insoluble aggregates by covalent or non-covalent interactions [34]. The result of solubility is similar to that of myofibrillar protein from *Culter alburnus* oxidized by hydroxyl radical [18], and also echoes the result of particle size distribution in this study.


**Table 2.** Solubility, WHC, OHC, and foaming property of WPI oxidized with different concentration of AAPH.

WPI = walnut protein isolates, AAPH = 2, 2- -azobis (2-amidinopropane) dihydrochloride; WHC = water holding capacity; OHC = oil holding capacity; FA = foaming ability; FS = foaming stability. Different letters within a column indicate significant differences (*p* < 0.05).

#### 3.3.2. Foaming Ability (FA) and Foaming Stability (FS)

Foaming property is an important interfacial property for protein processing, and is impacted by solubility, hydrophobicity, and flexibility of peptide chain. As indicated in Table 2, with the increase of AAPH concentration, the FA of WPI increased first and then decreased, and reached the maximum (36.67%) when the concentration of AAPH was 1 mmol/L. This is possibly because appropriate oxidation makes non-covalent bonds of WPI unfold, thereby the flexibility of the peptide chain increases. Consequently, more protein was adsorbed and formed a relatively stable gas–liquid interface. However, when WPI was over oxidized, the internal structure of the protein was largely unfolded and reconstructed, which led to the drop down of solubility markedly, and then a decline in FA. Meanwhile, FS also showed a similar trend by oxidation, and the maximum (83.33%) was identified at 1 mmol/L AAPH. The above research is similar to that on chickpea protein and rice bran globulin [23,24].

#### 3.3.3. Water Holding Capacity (WHC) and Oil Holding Capacity (OHC)

WHC and OHC are known to be linked to the water/oil binding ability of protein, and can significantly affect the texture of protein foods. As presented in Table 2, the WHC and OHC of oxidized WPI exhibited an initial increment followed by a decreasing tendency with AAPH concentration, and both the WHC (4.05 g/g) and OHC (5.84 g/g) reached maximum at 3 mmol/L AAPH. This is probably because, on the one hand, the hydrophobic groups of WPI were partly unfolded by oxidation, and some charged amino acid exposed to the surface, thus made the net charge of WPI increased [12]; and these mentioned changes of protein would be an advantage for WHC. On the other hand, macromolecular protein aggregation was formed by intermolecular disulfide bond crosslinking in oxidation [18], which would be a disadvantage for WHC. Therefore, the positive effects appear to be stronger than the negative effects in mild oxidation, and the water molecules entered the internal protein with non-covalent bonds, that made the WHC increase correspondingly. However, when the concentration was higher than 3 mmol/L AAPH, the negative effect was stronger than positive effects, thus decreasing the WHC dramatically. To the OHC, the oil adsorption capacity of WPI was enhanced with the hydrophobicity in certain concentrations of AAPH (≤3 mmol/L), and the OHC increased accordingly. Nevertheless, in strong oxidative conditions (>3 mmol/L), WPI was crosslinked and aggregated, which reduced the OHC consequently.

#### *3.4. Proteomic Analysis of Oxidative Modified WPI*

In order to further investigate the impact of oxidation on the structure of WPI, a Nano LC-MS/MS peptide sequencing analysis was performed, and the total ion chromatograms (TIC) of unoxidized and oxidized protein were shown in Figure 5. Although the trend of the TIC plot of both samples seemed to be very similar, the differences in protein abundance still revealed changes.

**Figure 5.** The total ion chromatogram (TIC) of WPI and oxidized WPI. WPI = walnut protein isolates.

By mass spectrometry analysis, a total of 3095 peptide spectra were detected, of which 646 protein groups were corresponding to 2396 unique peptide sequences (Supporting Information File S1, Table S1). According to their respective peptide sequences, the modifications in the functional group of the amino-acid side chains were analyzed and are listed in Table 3, including acetyl, carbamidomethyl, carboxyethylation, γ-glutamic semialdehydes (GGS), dehydrated, dehydroalanine, deoxidation, 4-hydroxy-2-nonenal (HNE), hydroxykynurenine, kynurenin, lactyllysine, malondialdehyde (MDA), oxidation, trioxidation, α-aminoadipic acid (AAA), and α-aminoadipic semialdehyde (AAS). For the different amino acids, cysteine, methionine, and tryptophan had the greatest number of side-chain modification types (5), followed by histidine, tyrosine and proline (4), and phenylalanine (3), whereas methionine and arginine created two types of modification. The above results are consistent with results previously reported on amino acids side-chains most prone to oxidative modification [35]. These modifications by oxidation may induce changes in the structure of the proteins, which in turn affected the function of proteins. For example, cysteine has unique reactivity which is associated with sulfhydryl reactive chemicals, hence, cysteine substitution has been a powerful tool to investigate the structure and function of proteins [36]. Methionine oxidation may influence the peptide hydrophobicity [37], where histidine residues in proteins are major targets for reaction with the lipid peroxidation product HNE [38], and so forth.

The UpSet plot and Venn diagram were constructed to further visualize the numbers and intersections of the identified peptide from different modifications. From Figure 6, the top five modifications ranked by modified number were carbamidomethyl, oxidation, HNE, MDA, and deoxidation. They were 243, 169, 64, 61, and 61 times, respectively. Of them, 124 times occurred alone for carbamidomethyl, and 119 times were the combination of carbamidomethyl with other modifications. The Wayne diagram in the upper part of the figure also showed the same results. It was generally believed that proteins from an animal source were more susceptible to undergoing oxidative reactions than plant proteins, while GGS and AAS were the significant protein oxidative indicators in meat and dairy products [39]. The results of this study indicated that numerous modifications also occurred in oxidized WPI, whereas carbamidomethyl and oxidation was the most significant indicator.


**Figure 6.** (**a**) UpSet plot of interactions between different modifications. (**b**) Venn diagram of interactions between different modifications. AAA = α-aminoadipic acid; AAS = α-aminoadipic semialdehyde; GGS = γ-glutamic semialdehydes; HNE = 4-hydroxy-2-nonenal; MDA = malondialdehyde.

To present the degree of modification on different protein groups more intuitively, a heatmap (Figure 7) was drawn in log10-transformed modification numbers (only total modification numbers equal to or greater than 5 are displayed). From Figure 7, oxidation, carbamidomethyl, deoxidation, HNE, and trioxidation were, in order, the most modified protein groups, and their modification numbers were greater than 5. This conclusion was basically consistent with the conclusion above. For different protein groups, the top 10 protein groups with the most modification numbers were P93 198 (190 times), A0A2I4F6R4 (127 times), A0A2I4DYF1 or Q9SEW4 (117 times), A0A2I4EG83 (108 times), Q2TPW5 (94 times), A0A2I4E5L6 (90 times), A0A2I4GEH1 (82 times), A0A2I4DYF1 (81 times), and Unknown (43 times). These proteins were mainly depicted as albumin seed storage protein, legumin-like protein, vicilin-like protein, and 11 S globulin seed storage protein. Interestingly, most of these proteins were found to be associated with allergies [40,41].

Mass spectrometry of these peptides detected the presence of 519 proteins, and detailed information of these proteins is presented in Supporting Information File S2: Table S2. To further understand the functions of these proteins, gene ontology (GO) annotation was performed (Supporting Information File S3: Table S3). Through analysis, 378 GO terms were annotated, including 115 terms in biological process (BP), 204 in molecular function (MF), and 59 in cellular component (CC), and the top 10 enriched GO terms of different classifications are displayed in Figure 8. It can be seen clearly that "cell redox homeostasis" was the most enriched (number = 31) in BP, while the most enriched (number = 46) in CC was "integral component of membrane". For MF, it was "ATP binding" with 33 enrichment numbers. Taking into consideration that protein modifications, especially post-translational modification, could regulate the structure and function of protein, including lifetime, assembly, localization, function, and degradation, it was therefore of great interest to further understand the main biological functions of these proteins [42].

**Figure 7.** The heatmap of modification numbers. AAA = α-aminoadipic acid; AAS = α-aminoadipic semialdehyde; GGS = γ-glutamic semialdehydes; HNE = 4-hydroxy-2-nonenal; MDA = malondialdehyde.

**Figure 8.** The top 10 functional enrichment analyses of gene ontology (GO).
