Joint technique “parallel peptide synthesis & de novo sequencing” development for the structure verification and high-throughput activity screening of biological peptides from sea cucumber (Stichopus japonicus) intestinal hydrolysate

This study introduces a novel joint technique combining "parallel peptide synthesis" and "de novo sequencing", which facilitates the high-throughput screening and structure validation of biological peptides derived from food and traditional Chinese medicine (TCM). Sea cucumber (Stichopus japonicus) was used as a model organism, undergoing simulated gastrointestinal digestion followed by de novo sequencing to predict potential peptides. Subsequently, cost-effective filter pipette tips were innovatively employed as parallel reaction vessels, enabling efficient peptide synthesis through the microfluidic flow of amino acid solutions over the loaded resin. After high-throughput biological activity screening, peptide YPGQLT was identified as the most potent antioxidant and acetylcholinesterase (AChE) inhibitor. It was then subjected to a molecular docking study to further explore its potential ligand-receptor interactions. This synergistic effect highlights peptide YPGQLT as a promising candidate for Alzheimer's disease (AD) treatment, thereby enhancing the potential biomedical application of sea cucumber. Notably, de novo predicted sequences can be further verified by comparing their characteristics, such as retention time and MS/MS spectrum, with those from the standard reference synthesized using this parallel synthesis protocol.