Dataset Information

Tetraspanin-based immunocapture for high-depth proteomic profiling of extracellular vesicles from cerebrospinal fluid for biomarker discovery

ABSTRACT: Due to its proximity to cells of the central nervous system, cerebrospinal fluid (CSF) is an important source of novel biomarkers for neurological diseases. Membrane-bound extracellular vesicles (EVs) are enriched for proteins of intracellular and membrane origin, implicated in the pathogenesis of some neurological diseases and secreted into CSF. Proteomic profiling of CSF-EVs, however, is limited by the large volumes required for typical EV isolation protocols. We appraised the performance of tetraspanin-based immunocapture to reduce required sample volume for EV isolation and subsequent proteomic profiling by library-free data independent-acquisition (DIA) mass spectrometry. Immunocapture was effective using CSF volumes as low as 200 µL, consistently detecting core EV markers and reducing relative levels of non-vesicular proteins such as Apolipoprotein B and galectin 3 binding protein (LGALS3BP) compared with size-exclusion chromatography (SEC). Proteomic depth reached 811±14 protein groups in EVs from 200 µL CSF, increasing to 1285±224 when using feature alignment across runs with up to 1000 µL starting volume. Increased depth was observed for both transmembrane and secreted proteins using immunocapture compared with SEC, with proportional enrichment of transmembrane proteins. Abundance and precursor peptide locations for potential neuronal-specific immunocapture targets described in the literature were assessed, including L1CAM and ATP1A3. This work demonstrates the effectiveness of tetraspanin immunocapture for proteomic profiling of EVs in small volumes of CSF that can be adapted to use with cell-type-specific markers of choice.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cerebrospinal Fluid

SUBMITTER: iolanda Vendrell

LAB HEAD: Alexander G Thompson

PROVIDER: PXD061653 | Pride | 2026-02-23

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	EV1000_report.pg_matrix.tsv	Tabular
	EV200_report.pg_matrix.tsv	Tabular
	EV500_report.pg_matrix.tsv	Tabular
	EVSEC_report.pg_matrix.tsv	Tabular
	Fullset_report.pg_matrix.tsv	Tabular

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Tetraspanin-based immunocapture for high-depth proteomic profiling of extracellular vesicles from cerebrospinal fluid for biomarker discovery.

Dellar Elizabeth R ER Vendrell Iolanda I Fischer Roman R Thompson Alexander G AG

Clinical proteomics 20260117 1

<h4>Background</h4>Due to its proximity to cells of the central nervous system, cerebrospinal fluid (CSF) is an important source of novel biomarkers for neurological diseases. Membrane-bound extracellular vesicles (EVs) are enriched for proteins of intracellular and membrane origin, implicated in the pathogenesis of some neurological diseases, and secreted into CSF. Proteomic profiling of CSF-EVs, however, is limited by the large volumes required for typical EV isolation protocols.<h4>Methods</h ...[more]

PMID: 41547780

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Project description:Introduction: Cell-derived extracellular vesicles ([EVs], i.e., exosomes and microparticles) have been reported to mediate the cardioprotective effects of stem cells. However, a head-to-head comparison of their effects with those of the cells from which they derive has not been reported. Hypothesis: The effects of the EV content of human embryonic stem cell (hESC)-derived cardiac progenitors may be equivalent to those of the parent cells. Methods: Two series of immunodeficient (nude) mice underwent permanent coronary artery ligation. Three weeks later, those with an echocardiographically-determined LV ejection fraction ≤ 50% were randomly allocated to receive transcutaneous echo-guided peri-infarct injections of alpha-MEM media (controls), hESC-derived SSEA-1+ cardiac progenitors (500000 cells in 30 μL), or total EV secreted by those 500000 cells in 48 hours in an equivalent volume. The second series also included sham-operated animals for which the needle was inserted into the myocardium in three places without injection. EVs were collected from the cell-secreted medium by ultracentrifugation and characterized by flow cytometry, and nanosight NTS. Outcomes were assessed after 6 weeks by echocardiography, taking LV end-systolic volume (ESV) as the primary end point. Hearts were processed for the assessment of fibrosis and angiogenesis. Total RNA was extracted from carefully selected animals for gene expression analysis by Affymetrix chip. Only animals with similar VTS at baseline were used for this analysis. All data were collected and analyzed blindly. Results: Cardiac progenitors were successfully generated by exposure of the pluripotent ESC to bone morphogenetic protein-2, purified by anti-CD15 immunomagnetic sorting and then cultured on vitronectin for 48 hours, after which cells or EVs derived from the same cell batch were injected. After 6 weeks in the first series, LVESV [m±SEM] in controls did not significantly differ from the pre-injection value (difference: -2.64 ± 1.54 µL, p=0.12, n= 12). Conversely, in the cell-treated group, LVESV significantly decreased by -4.20 ± 0.96 µL (p=0.0007, n=16). A similar decrease was seen in the EV-treated group: -5.73 ± 1.21 µL (p=0.0003, n=15). Similar patterns were seen for LV end-diastolic volumes: controls (-2.46 ± 1.19 µL, p=NS), cells (-4.48 ± 1.47 µL, p=0.009), EV (-4.29 ± 1.31 µL, p=0.005). For the second series, the VTD of cell- and EV-treated animals remained stable (cell: -1.85 ± 3.27 µL, p=NS, n= 4; EV: +0.05 ± 1.53 µL, p=NS, n = 4), whereas sham- and control-treated animals deteriorated significantly (sham: +2.20 ± 0.35 µL, p = 0.003, n = 5; control: +3.27 ± 0.87 µL, p = 0.03, n = 4). The trend held for VTS, though differences were not significant. Analysis of gene expression data revealed interesting pathways that were more highly expressed in cell- and EV-treated animals than in controls and sham-operated animals. Conclusions: These data support the hypothesis that EVs may be critical mediators of the paracrine effects of cell therapy, and for the sake of streamlining translational processes, deserve to be considered as potential alternatives to stem cell transplantation.

Project description:Alzheimer’s disease (AD) is a major neurodegenerative disease characterized by extracellular amyloid β (Aβ) plaques and intracellular hyperphosphorylated tau (P-tau). Increasing evidence indicates that extracellular vesicles (EVs) play an important role in AD pathogenesis. We previously reported that the choroid plexus epithelial (CPE) cells, present at the interface between blood and cerebrospinal fluid (CSF), show increased secretion of EVs into the CSF in response to peripheral inflammation. Here, we studied the importance of CP-derived EVs in AD pathogenesis. We observed increased EV levels in the CSF of 7 weeks old transgenic APP/PS1 mice, correlating with higher Aβ CSF levels at this age, while levels normalized with age. To study whether the elevated Aβ CSF levels might be responsible for this increase, mice were intracerebroventricularly (icv) injected with Aβ oligomers (AβO) which revealed a significant increase in the amount of CD9 and CD81 positive EVs in the CSF. The importance of the CPE cells as EV source was shown by in vitro analysis of AβO stimulated primary CPE cells and in vivo analysis of the CP by transmission electron microscopy (TEM). Evaluation of EV marker immunostaining, both from AβO icv injected mice and APP/PS1 mice, confirmed the upregulation of EV biogenesis in the CP. Interestingly, AβO-induced, CP-derived EVs induced pro-inflammatory effects in mixed cortical cultures (MCC) whereas proteome analysis revealed the presence of complement protein C3, amongst other inflammatory proteins. Additionally, we could show for the first time that AβO induce an upregulation of C3 in CPE cells. These data highlight the CP and CP-derived, C3-containing EVs as novel players in the emerging importance of the complement pathway in the pathogenesis of AD. Strikingly, inhibition of EV production using GW4869 resulted in protection against the AβO-induced cognitive decline. In conclusion, our results show that intraventricular AβO induce both C3 activation and EV secretion by the CP and that these EVs contain pro-inflammatory molecules, including C3, which play a role in neuroinflammation and loss of cognition. This suggests that inhibition of EV production by the CP might be an interesting therapeutic approach to be explored in the prevention or treatment of AD.