A Molecular ID Card for Circulating Exosomes

Small extracellular vesicles (sEVs, which include exosomes) are tiny membrane-bound packages, roughly 30 to 150 nm across, that cells release into the bloodstream. They carry proteins, lipids and nucleic acids between cells, and hold enormous potential as disease biomarkers and therapeutic delivery vehicles. But a persistent problem has dogged the field: how do you know you're actually studying sEVs and not the lipoprotein particles and protein aggregates that outnumber them in plasma by a million to one?

A study published in Nature Cell Biology (November 2025) by researchers at the Baker Heart and Diabetes Institute now provides an answer. Analysing over 140 human plasma samples with high-resolution density gradient separation and multi-omics profiling, the team identified 182 core proteins and 52 core lipids that universally define authentic circulating sEVs.

Why existing markers weren't good enough

Most sEV markers in common use were developed in cell culture. When the researchers tested eleven of these against actual human plasma, only two (CD9 and HSPA8) showed up consistently. That disconnect has been muddying results across the field, making it hard to compare studies, validate isolation methods, or trust biomarker discoveries built on potentially impure starting material.

The headline findings

From 4,631 proteins detected in purified plasma sEVs, the team distilled 182 that were consistently enriched across individuals and sEV subpopulations. Of these, 151 are surface-accessible, including familiar names like CD81, CD63, CD9 and CD44 alongside integrins and annexins. On the lipid side, 52 species stood out, with phosphatidylserine and phosphatidylinositol phosphates among the most distinctive.

Two markers emerged as the top validators. ADAM10, a metalloprotease, was found exclusively in sEV fractions at up to 7.5-fold higher abundance, retained enzymatic activity on vesicle surfaces, and proved universally conserved. PS(36:1), a phosphatidylserine species, was the leading lipid marker, and its ratio to the lipoprotein-associated cholesterol ester CE(18:0) reliably separated genuine sEVs from contaminants. Using machine learning, the team achieved 97% accuracy in telling authentic sEVs apart from non-EV particles.

Proof in a clinical cohort

To show practical value, the researchers applied their marker panel to an early coronary artery disease cohort. All 182 core protein markers remained stable between diseased and healthy groups, confirming that the sEV signature holds regardless of disease state. Within those authenticated preparations, they then identified over 200 differentially expressed proteins tied to coronary artery calcification, including cystatin C and thioredoxin reductase 2.

What this means for the field

This study gives the EV community something it has long needed: a validated reference standard. Researchers can now benchmark isolation protocols against the 182/52 marker signature, use ADAM10 and PS(36:1) for rapid quality control, flag lipoprotein contamination using exclusion markers like CE(18:0), and compare results across laboratories with a shared molecular framework. The complete datasets are freely available through EVmap.

One caveat worth noting: the sEVs isolated here contained both exosomes (endosomal origin) and microvesicles (shed from the plasma membrane). Teasing apart which markers are specific to each subtype remains an open question for future work.

Reference: Rai, A., Huynh, K., Cross, J. et al. Multi-omics identify hallmark protein and lipid features of small extracellular vesicles circulating in human plasma. Nat Cell Biol 27, 2167-2185 (2025). https://doi.org/10.1038/s41556-025-01795-7

IMAGE SEV CHarcteristics CREDIT Rai et al

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