Toward Identification of Markers for Brain-Derived Extracellular Vesicles in Cerebrospinal Fluid: A Large-Scale, Unbiased Analysis Using Proximity Extension Assays

Abstract

Extracellular vesicles (EVs) captured in biofluids have opened a new frontier for liquid biopsies. To enrich for vesicles coming from a particular cell type or tumour, scientists utilize antibodies to transmembrane proteins that are relatively unique to the cell type of interest. However, recent evidence has called into question the basic assumption that all transmembrane proteins measured in biofluids are, in fact, EV-associated. To identify both candidate markers for brain-derived EV immunocapture and cargo proteins to validate the EVs' cell of origin, we conducted an unbiased Olink screen, measuring 5416 unique proteins in cerebrospinal fluid after size exclusion chromatography. We identified proteins that demonstrated a clear EV fractionation pattern and created a searchable dataset of candidate EV-associated markers-both proteins that are cell type-specific within the brain, and proteins found across multiple cell types for use as general EV markers. We further implemented the DeepTMHMM deep learning model to differentiate predicted cytosolic, transmembrane, and external proteins and found that intriguingly, only 10% of the predicted transmembrane proteins have a clear EV fractionation pattern based on our stringent criteria. This dataset further bolsters the critical importance of verifying EV association of candidate proteins using methods such as size exclusion chromatography before downstream use of the targets for EV analysis.

Keywords: Olink; brain‐derived extracellular vesicles; membrane association; proteomics; size exclusion chromatography.

FIGURE 1

CSF SEC fractionation as a measure of EV association. (a) Quantification of CSF fractions using a Simoa assay for CD81. Four individual healthy CSF samples were fractionated using SEC, and each fraction was analysed by Simoa. A Mann–Whitney U test performed comparing fractions 9 and 10 with fractions 7, 11, 12 and 13 in all samples combined showed fractions 9 and 10 are significantly greater than fractions 7, 11, 12 and 13 (p < 0.0005). (b) Quantification of CSF fractions using the Olink assay for CD63. Four individual healthy samples were fractionated using SEC, and each fraction was analysed by the Olink HT panel. The Mann–Whitney U test performed comparing fractions 9 and 10 with fractions 7, 11, 12 and 13 in all samples combined showed fractions 9 and 10 are significantly greater than fractions 7, 11, 12 and 13 (p < 0.0005). (c) Heat maps showing normalized NPX values for each SEC fraction for four representative previously published EV contaminants and four EV‐associated proteins in the Olink panel. Of note, the EV contaminant proteins F2, C3, FN1 and SERPINF1 (PEDF) all have increasingly high NPX values predominantly in the late free protein fractions. EV‐associated proteins ANXA2, ANXA4, ANXA5 and VTA1 show EV‐associated fractionation patterns with high NPX values in fractions 9 and 10. Anxa5 and VTA1 also show NPX signals in later fractions 14 and 15, suggesting possibly soluble protein isoforms for these proteins. (d) Percentage of Deep TMHMM predicted transmembrane, internal, and external targets quantified by Olink as having an EV fractionation pattern in CSF. CSF, cerebrospinal fluid; NPX, normalized protein expression; PEDF, pigment epithelium‐derived factor; SEC, size exclusion chromatography.

FIGURE 2

Cell‐type‐specificity of proteins that show an EV‐associated fractionation pattern. The EV Association Score (EV‐associated NPX signals in fractions 9 and 10 were greater than NPX signals in fractions 7, 11, 12 and 13) and calculated Tau Score of > 0.75 for each identified transmembrane (red), internal (blue) and external (green) proteins that demonstrated an EV‐associated fractionation pattern for (a) astrocytes, (b) endothelial cells, (c) microglia, (d) oligodendrocytes, and (e) neurons. NPX, normalized protein expression.