*Article* **Effect of Peroxyl Radical-Induced Oxidation on Functional and Structural Characteristics of Walnut Protein Isolates Revealed by High-Resolution Mass Spectrometry**

**Xuechun Zhang 1,2,3,4, Xi Yang 4, Yunqian Li 4, Zhenxing Wang 2, Xuemei He 1,\* and Jian Sun 3,\***


**Abstract:** The present study aims to investigate the structural and functional properties of oxidated walnut protein isolates (WPI) by 2,2- -azobis (2-amidinopropane) dihydrochloride (AAPH). The oxidation degree, changes in structural characteristics, processing properties, and protein modifications of WPI were measured. The results showed that oxidation significantly induced structural changes, mainly reflected by the increasing carbonyl content, and decreasing sulfhydryl and free amino groups. Moreover, the secondary structure of WPI was altered in response to oxidation, and large aggregates formed through disulfide cross-linking and hydrophobic interactions. Almost all the property indicators were significantly decreased by oxidation except the foaming property and water/oil holding capacity. Mass spectrometry analysis showed that 16 different modifications occurred in amino acid side chains, and most of the protein groups with higher numbers of modifications were found to be associated with allergies, which was further confirmed by the reduction in antigenicity of the major allergen (Jug r 1) in WPI. Meanwhile, we used oxidation-related proteins for gene ontology (GO) enrichment analyses, and the results indicated that 115, 204 and 59 GO terms were enriched in terms of biological process, molecular function, and cellular component, respectively. In conclusion, oxidation altered the groups and conformation of WPI, which in turn caused modification in the functional properties correspondingly. These findings might provide a reference for processing and storage of walnut protein foods.

**Keywords:** walnut protein isolates; oxidation; structural characteristics; functional properties; proteomics
