Subtype-Specific Enrichment of HER2-Positive Extracellular Vesicles Enables Metabolomic Profiling for Breast Cancer Liquid Biopsy

Extracellular vesicles have emerged as crucial components in the advancement of liquid biopsy. However, the efficient isolation of molecularly defined subpopulations remains challenging, limiting the performance of liquid chromatography−tandem mass spectrometry (LC−MS/MS)-based metabolomic analysis. In this study, HER2-targeting peptides were identified via phage display screening against the extracellular domain of HER2 and used to construct a peptide-directed magnetic separation platform (SA@HER2-2-MPMs) for the rapid and selective enrichment of HER2-positive extracellular vesicles. The biotinylated peptide enabled the specific recognition of HER2-enriched EVs’ membranes, while streptavidin-functionalized magnetic polystyrene microspheres facilitated their efficient capture and magnetic separation. The enriched extracellular vesicles were subjected to subsequent metabolite extraction and LC−MS/MS analysis, forming the basis of an integrated workflow for subtype-specific extracellular vesicles isolation and metabolomic profiling. Compared with UC preparations, the peptide-directed workflow yielded lower measured concentrations of selected plasma-associated contaminants and supported downstream LC−MS/MS profiling. Metabolomic analysis revealed disease-associated alterations, and an exploratory predictive reanalysis using nested cross-validation and a held-out internal subset showed high within-cohort discrimination. This study establishes a peptide-directed strategy for isolating molecularly defined EV subpopulations and provides a platform for exploratory metabolic biomarker discovery in HER2-positive breast cancer; independent external validation remains required.