LC-MS/MS-based peptide identification, molecular simulations, and biological validation for targeted discovery of...

Food-derived antioxidant peptides are promising alternatives to synthetic antioxidants, but targeted discovery remains challenging. Here, activity-guided fractionation, LC-MS/MS-based peptide identification, molecular simulations, and biological validation were integrated to identify antioxidant peptides from large yellow croaker protein hydrolysates. LPH-3 and its F3 sub-fraction exhibited the highest antioxidant activities, and 89 peptides were identified in F3. After in silico screening, 16 candidates were docked against Keap1, from which SG-8, DN-11, and GR-13 were selected for synthesis. SG-8 showed the strongest radical-scavenging and cytoprotective activities in H₂O₂-injured Caco-2 cells. Three independent 500 ns molecular dynamics simulations per complex indicated smaller conformational deviations and more favorable estimated interaction energetics for the modeled Keap1-SG-8 complex, consistent with the experimental activity ranking. RT-qPCR and ML385 inhibition supported the involvement of Nrf2-related antioxidant signaling, although cytoplasmic availability and direct Keap1-SG-8 binding remain unverified. Overall, SG-8 is a promising marine-derived antioxidant peptide warranting further development and mechanistic validation.