# Development of Endogenous Protein Probes for Characterizing Surface Proteins and Cellular Interactors of Extracellular Vesicles

## TL;DR

A new genetic tool uses APEX2 to label and analyze proteins on extracellular vesicles, helping to better understand their role in cell communication and biomarker discovery.

## Contribution

A genetically encoded proximity labeling probe for high-fidelity mapping of EV surface proteins and interactors.

## Key findings

- APEX2 fusion with EV-sorting scaffolds enables biotinylation of EV surface proteins and corona components.
- Streptavidin enrichment and mass spectrometry reveal subpopulation-specific surfaceome and interactome.
- The method provides a robust framework for EV biology and biomarker discovery.

## Abstract

Extracellular vesicle (EV) surface proteins, derived from producer cells and their surrounding environment, represent a valuable source of biomarkers and participate in a plethora of biological functions, including intercellular communication. However, current methods struggle to distinguish core EV surface proteins from adsorbed corona proteins or map the EV‐cell interplay. Here, a genetically encoded proximity labelling probe is presented that displays engineered ascorbate peroxidase, APEX2, on the surface of EVs via fusion to EV‐sorting scaffold proteins. This enables the biotinylation of producer‐cell‐derived surface proteins, corona proteins, and interactors in vitro. After the enrichment by streptavidin bead pulldowns, subpopulation‐specific, biotinylated surfaceome and interactome are comprehensively characterized using mass spectrometry‐based proteomics. Thus, a genetic tool is introduced for the high‐fidelity mapping of the surfaceome and cellular interactome of EVs in vitro. This approach offers a robust framework for dissecting EV biology and has broad applications in biomarker discovery and EV‐based therapeutics.

The proximity labeling enzyme APEX2 is displayed on extracellular vesicle (EV) surfaces via genetic fusion with EV‐sorting scaffolds, enabling in situ biotinylation of native surface proteins, adsorbed corona components, and interacting cellular proteins. Streptavidin enrichment coupled with mass spectrometry reveals subpopulation‐specific surfaceome and interactome, providing a precise platform for biomarker discovery and mechanistic dissection of EV‐mediated communication.