Integrated proteomics and in silico analysis of casein-derived peptides with antioxidant, antihypertensive, and anticancer activities

Bioactive peptides (BAPs) from milk proteins hold promise for functional foods and therapeutics due to their diverse biological activities and low toxicity. This study identified novel casein-derived peptides using simulated gastrointestinal digestion, proteomics, and in-silico prediction tools. High-resolution tandem mass spectrometry and computational screening led to the identification of three novel peptides: FVAPFPEVFG (αCN) , NLLRF (αCN) , and EMPFPKY (βCN) . Chemical and cellular assessments revealed distinct bioactive profiles, with EMPFPKY (βCN) demonstrating superior antioxidant activity through Keap1 binding, while FVAPFPEVFG (αCN) exhibited dual ACE-inhibitory and anticancer properties against MCF-7 breast cancer cells. Furthermore, the selective cytotoxicity of NLLRF (αCN) against MCF-7 cancer cells, while preserving the viability of normal cells, suggested its potential for therapeutic applications. This comprehensive approach, combining experimental and computational methods, establishes a robust pipeline for identifying and characterizing bioactive peptides, contributing to the development of novel functional food products and pharmaceutical applications. • Novel milk peptides identified with distinct functional bioactivities. • EMPFPKY (β-CN) reduces oxidative stress via Nrf2-linked radical scavenging. • FVAPFPEVFG (α-CN) shows ACE inhibition with strong binding affinity. • NLLRF (α-CN) and FVAPFPEVFG (α-CN) target MCF-7 cells sparing normal cells. • Computational and experimental integration streamlines peptide discovery.