Proximity Proteomics Has Potential for Extracellular Vesicle Identification

Extracellular vesicles (EVs) are biomarkers and mediators of intercellular communication. In biological samples, EVs are secreted by various types of cells. The proteomic identification of proteins expressed in EVs has potential to contribute to research and clinical applications, particularly for cancer. In this study, researchers from the Saitama Medical University used the proximity-labeling method-based proteomic approach for EV identification, labeling membrane components proximal to a given molecule on the EV membrane surface. Due to the small labeling range, proteins on the surface of the same EVs are likely to be labeled by selecting a given EV surface antigen. The protein group of cancer cell-secreted EV (cEV), which abundantly expresses a close homologue of L1 (CHL1), was examined using a model mouse for lung cancer (LC). cEV-expressed proteins were identified by proteomic analysis of enzyme-mediated activation of radical sources by comparing serum EVs from wild-type and LC mice. SLC4A1 was found to be co-expressed in CHL1-expressing EVs, highlighting EVs expressing both CHL1 and SLC4A1 as candidates for cEVs. Serum EVs expressing both CHL1 and caspase 14 were significantly elevated in LC patients compared with healthy individuals. Thus, the combination of proximity labeling and proteomic analysis allows for effective EV identification.

EMARS reaction

Schematic illustration of the EMARS reaction using fluorescein-tyramide (FT). HRP-conjugated cognitive molecules (e.g., antibodies) recognizing the given molecule were prepared (“EMARS probe”) and added to living cells, followed by treatment with FT. After the EMARS reaction, the sample proteins (“EMARS products”) were subjected to gel electrophoresis and/or mass spectrometry analysis for the purification of fluorescein-labeled proteins as proximity molecules.