Unraveling the paracellular absorption mechanism of egg-derived peptide RVPSL mediated by cytoskeleton and tight junction phosphorylation via 4D-FastDIA phosphoproteomics.

The egg white-derived peptide RVPSL possesses antihypertensive activity. However, its oral bioavailability is limited primarily by low paracellular absorption efficiency. This study aimed to elucidate the molecular mechanism underlying RVPSL absorption, with a focus on the regulation of phosphorylation. Using the Caco-2 monolayer model integrated with 4D-FastDIA quantitative phosphoproteomics, bioinformatics, and parallel reaction monitoring (PRM), changes in phosphorylation during RVPSL absorption were systematically analyzed. Significant alterations in the phosphorylation status of cytoskeletal and tight junction (TJ)-associated proteins were observed. Site-specific phosphorylation changes were identified in key targets including filamin A (FLNA), focal adhesion kinase (FAK), Src, vinculin, zonula occludens-1 (ZO-1), and ZO-2. Specifically, increased phosphorylation of FLNA at Ser2152, along with changes at key sites on FAK and Src, promoted reorganization of the actin cytoskeleton, thereby facilitating the dynamic opening of the paracellular space. Concurrent phosphorylation of ZO-1 and ZO-2 contributed to the dynamic regulation of TJ integrity. These coordinated phosphorylation events enhanced epithelial paracellular permeability, thereby facilitating RVPSL absorption via the paracellular pathway. Overall, this study reveals the phosphorylation-mediated regulation of paracellular absorption for RVPSL, offering new insights into the absorption mechanism of egg white-derived bioactive peptides.