Engineering Peptide-Based Molecular Baits for Targeted\nFishing and Protein Profiling of Exosomes as a Liquid Biopsy for Gastrointestinal\nAdenocarcinoma

High-performance isolation of exosomes\nas a promising liquid biopsy\ntarget is of great importance for both fundamental research and clinical\napplications. This is, however, challenged by the prevalent heterogeneity\nof exosomes and the highly complex nature of biosamples. Here, we\nintroduce the use of a CD81-targeting peptide as a building block\nfor tailoring molecular baits for exosome isolation and payload analysis\nin clinical biofluids. To explore the full potential of multivalent\ninteractions, peptide-functionalized affinity interfaces were covalently\nengineered with varied assembling topology, flexibility, and local\ndensity of the recognition motif. Capable of best fitting the surface\nconformation of CD81 on highly curved exosome membranes, a dual-layered\nexosome capture affinity interface (Exo-PepTrap2) with tandem bivalent\npeptide decoration outperforms the monolayered and the branched multivalent\narchitectures. Enabled by the multivalency-enhanced affinity reaction\nand antifouling ability, Exo-PepTrap2 achieved a high yield and purity\nfor targeted fishing of exosomes in complex cell culture media and\nclinical urine samples. By integration of Exo-PepTrap2 isolation with\nmass spectrometry-based proteomic profiling, differentially expressed\nproteins were efficiently identified in harvested exosomes as potential\nbiomarkers for gastrointestinal adenocarcinoma. This CD81-targeted\ntandem peptide-functionalized affinity platform provides a new viewpoint\nfor tailoring multivalency-based affinity interfaces and a versatile\ntool to explore molecular information in exosomes for precise medicine.