Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes

TLDR

Extracellular vesicles are diverse in size and origin, yet specific markers for distinct subpopulations are lacking, limiting functional studies. EVs from human dendritic cells were fractionated by sedimentation speed followed by iodixanol gradient floatation or immuno‑isolation. Quantitative proteomics revealed that traditional exosome markers are ubiquitous across EVs, while distinct protein signatures enriched in small EVs were identified, leading to a set of five categories and the proposal of immuno‑isolation using CD63, CD81, or CD9 to separate exosomal and nonexosomal subpopulations.

Abstract

Extracellular vesicles (EVs) have become the focus of rising interest because of their numerous functions in physiology and pathology. Cells release heterogeneous vesicles of different sizes and intracellular origins, including small EVs formed inside endosomal compartments (i.e., exosomes) and EVs of various sizes budding from the plasma membrane. Specific markers for the analysis and isolation of different EV populations are missing, imposing important limitations to understanding EV functions. Here, EVs from human dendritic cells were first separated by their sedimentation speed, and then either by their behavior upon upward floatation into iodixanol gradients or by immuno-isolation. Extensive quantitative proteomic analysis allowing comparison of the isolated populations showed that several classically used exosome markers, like major histocompatibility complex, flotillin, and heat-shock 70-kDa proteins, are similarly present in all EVs. We identified proteins specifically enriched in small EVs, and define a set of five protein categories displaying different relative abundance in distinct EV populations. We demonstrate the presence of exosomal and nonexosomal subpopulations within small EVs, and propose their differential separation by immuno-isolation using either CD63, CD81, or CD9. Our work thus provides guidelines to define subtypes of EVs for future functional studies.