Orthogonal Coding-Guided Liposomal Nanoreactor for One-Pot Precise Proteomic Profiling of Tumor-Derived Extracellular Vesicle Subpopulations

The protein cargoes of tumor-derived extracellular vesicles (TEVs) are promising biomarkers for cancer diagnosis, yet their application is constrained by EV heterogeneity and complex assay procedures. Here, we developed an orthogonal coding-guided liposomal enzyme nanoreactor (OLEN) for the one-pot proteomic profiling of TEVs. It integrates a DNA computation module to selectively recognize CD63⁺EpCAM⁺ TEVs via dual-marker orthogonal coding. Simultaneously, enzymatic nanoreactors are delivered into the vesicles through targeted membrane fusion, enabling <i>in situ</i> protein extraction and digestion for streamlined proteomic analysis. Using only 10 μL of plasma from prostate cancer (PCa) patients, OLEN identified 525 proteins, covering over 86.5% EV proteins, with 81.7% annotated as tumor-associated. More than 200 proteins were differentially expressed between PCa and benign prostatic hyperplasia (BPH), with several upregulated proteins previously implicated with PCa progression. Its modular design, streamlined workflow, and minimal sample requirements make OLEN a promising platform for EV-based liquid biopsy and noninvasive cancer diagnostics.