High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array.

Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.