Engineering Peptide-Based Molecular Baits for Targeted Fishing and Protein Profiling of Exosomes as a Liquid Biopsy for Gastrointestinal Adenocarcinoma

High-performance isolation of exosomes as a promising liquid biopsy target is of great importance for both fundamental research and clinical applications. This is, however, challenged by the prevalent heterogeneity of exosomes and the highly complex nature of biosamples. Here, we introduce the use of a CD81-targeting peptide as a building block for tailoring molecular baits for exosome isolation and payload analysis in clinical biofluids. To explore the full potential of multivalent interactions, peptide-functionalized affinity interfaces were covalently engineered with varied assembling topology, flexibility, and local density of the recognition motif. Capable of best fitting the surface conformation of CD81 on highly curved exosome membranes, a dual-layered exosome capture affinity interface (Exo-PepTrap2) with tandem bivalent peptide decoration outperforms the monolayered and the branched multivalent architectures. Enabled by the multivalency-enhanced affinity reaction and antifouling ability, Exo-PepTrap2 achieved a high yield and purity for targeted fishing of exosomes in complex cell culture media and clinical urine samples. By integration of Exo-PepTrap2 isolation with mass spectrometry-based proteomic profiling, differentially expressed proteins were efficiently identified in harvested exosomes as potential biomarkers for gastrointestinal adenocarcinoma. This CD81-targeted tandem peptide-functionalized affinity platform provides a new viewpoint for tailoring multivalency-based affinity interfaces and a versatile tool to explore molecular information in exosomes for precise medicine.