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Automated enrichment and co-extraction pipeline for efficient analysis of urinary extracellular vesicle protein and RNA

ABSTRACT: Extracellular vesicles (EVs) cargoes have great potential as promising biomarkers for disease diagnosis. However, traditional methods for EV enrichment and cargo extraction are inefficient, limiting the accessibility of valuable biological information. Here, we present a bead-based pipeline, designed to efficiently enrich EVs from urine while simultaneously extracting both EV protein and RNA. Compared to traditional methods involving ultracentrifugation (UC) for EV isolation, acetone precipita-tion for protein extraction, and TRIzol for RNA extraction, the new pipeline achieved over a 5-fold improvement in EV protein and RNA yield. Additionally, it reduced the whole duration from approximately 2 days to just 1 h. When applied to the non-invasive diagnosis of prostate cancer (PCa), the pipeline demonstrated excellent diagnostic performance, with a panel of EV protein and RNA markers achieving an AUC of 0.82. This efficient and streamlined pipeline provides a robust tool for com-prehensive EV cargo analysis and holds great potential for advancing non-invasive diagnostics in clinical settings.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

DISEASE(S): Prostate Adenocarcinoma

SUBMITTER: Yufeng Liu

LAB HEAD: Ying Zhang

PROVIDER: PXD060304 | Pride | 2025-04-11

REPOSITORIES: Pride

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Project description:Circulating extracellular vesicles (EVs) contain molecular footprints - lipids, proteins, metabolites, RNA, and DNA - from their cell of origin and may provide non-invasive access for detection, characterization, and monitoring of diseases. Consequently, EV-associated RNA and proteins have gained widespread interest in developing liquid-biopsy assays for cancer. Yet, an integrative proteo-transcriptomic landscape of EVs and a direct comparison with their cell of origin remains relatively unexplored. Here, we report that EVs enrich for distinct molecular cargo and their proteo-transcriptome does not linearly correlate with their cell of origin. Our sub-cellular compartment analyses revealed that EV enrich 2-6 times more cytoskeletal and extracellular proteins, while their donor cells cargo 3-10 times more nuclear and mitochondrial proteins. EVs predominantly enrich (40-60%) for small RNA between ~15-200 nucleotides, which include miRNAs, siRNAs, tRNA, 5S rRNAs, and snRNAs, associated with cell differentiation, development, and Wnt signaling signatures. Finally, our integrated proteo-transcriptomic analyses reveal that EVs are enriched with specific small RNAs (RNY3, vtRNA, and MIRLET-7) and their complementary proteins (YBX1, IGF2BP2, SRSF1/2). These analyses suggest that RNA-protein complexes may constitute a functional interaction network inside EVs to protect and regulate access to EV-RNA. To ensure that our analyses are unbiased and independent of the cell of origin, EV isolation methods, site of experimentation, site of data generation, and methodologies applied for RNAseq and mass spectrometry, we have generated and curated comprehensive datasets of proteomics and transcriptomics from a total of 12 cancer cell lines, their EVs, and prostate cancer patientsÕ serum EVs (A detailed description is provided in Fig. 1). These combined analyses demonstrated that irrespective of the cell of origin and other aforementioned conditions, EVs enrich for distinct RNA and protein signatures that do not linearly correlate with their cell of origin.

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Automated enrichment and co-extraction pipeline for efficient analysis of urinary extracellular vesicle protein and RNA

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