Sequencing-Based Protein Analysis of Single Extracellular Vesicles

Circulating extracellular vesicles (EVs)-biological nanomaterials shed from most mammalian cells. have emerged as promising biomarkers, drug delivery vesicles, and treatment modulators. While different types of vesicles are being explored for these applications, it is becoming clear that human EVs are quite heterogeneous even in homogeneous or monoclonal cell populations. Since it is the surface EV protein composition that will largely dictate their biological behavior, high-throughput single EV profiling methods are needed to better define EV subpopulations. Here, we present an antibody-based immunosequencing method that allows multiplexed measurement of protein molecules from individual nanometer-sized EVs. We use droplet microfluidics to compartmentalize and barcode individual EVs. The barcodes/antibody-DNA are then sequenced to determine protein composition. Using this highly sensitive technology, we detected specific proteins at the single EV level. We expect that this technology can be further adapted for multiplexed protein analysis of any nanoparticle.

Automated summary

Circulating extracellular vesicles (EVs) are promising biomarkers and drug delivery vehicles, but human EVs exhibit significant heterogeneity even in homogeneous cell populations. An antibody-based immunosequencing method enables multiplex measurement of protein molecules from individual EVs, providing a way to detect specific proteins at the single EV level. This technology has the potential to be adapted for multiplexed protein analysis of any nanoparticle.