Site-specific N-glycoproteomics reveals alteration of N-glycan structures and heterogeneity to enhance thermally induced gelation characteristics of egg white proteins.

N-glycans are complex carbohydrates crucial for the structural integrity and functionality of proteins. Here, we present a high-resolution egg white protein N-glycoproteomic atlas that systematically unveils the microheterogeneity across N-glycosites, glycoproteins, and co-occurring N-glycans. Notably, hawthorn leaf flavonoids (HF) markedly reduced undecorated and high-mannose N-glycans while increasing paucimannose and sialylated-only N-glycans, and these changes strongly correlated with elevated protein stability. Site-specific N-glycan comparative analysis further revealed that HF increased 15 N-glycans at four glycoproteins (such as Hex9GlcNAc2 in ovotransferrin N492) and reduced 3 N-glycans at three glycoproteins (such as Hex3HexNAc6 in mucin-5B N69), promoting the formation of more ionic bonds, hydrophobic interactions, and hydrogen bonds, thereby converting the secondary structure from random coil to α-helix and β-sheet and ultimately forming a more tightly ordered egg white gel. Overall, this study provides a novel perspective on improving gel properties by modulating the structure and site-specific N-glycosylation of egg white proteins (EWPs).