Dataset Information

Plasma-derived EV Phosphoproteomics through Chemical Affinity Purification

ABSTRACT: Invasive nature and pain caused to patients inhibit the routine use of tissue biopsy-based procedures for cancer diagnosis and surveillance. The analysis of extracellular vesicles (EVs) from biofluids have recently gained significant traction in the liquid biopsy field. EVs offer an essential “snapshot” of their precursor cells in real time and contain information-rich collection of nucleic acids, proteins, lipids, etc. The analysis of protein phosphorylation, as a direct marker of cellular signaling and disease progression, could be an important stepstone to successful liquid biopsy applications. Here, we introduce a rapid EV isolation method based on chemical affinity called EVtrap (Extracellular Vesicles Total Recovery and Purification) for EV phosphoproteomics analysis of human plasma. Incorporating EVtrap with high performance mass spectrometry (MS), we were able to identify over 16,000 unique peptides representing 2,238 unique EV proteins from just 5 μL plasma sample, including most known EV markers, with substantially higher recovery levels compared to ultracentrifugation. Most importantly, more than 5,500 unique phosphopeptides representing almost 1,600 phosphoproteins in EVs were identified using only 1 mL of plasma. Finally, we carried out quantitative EV phosphoproteomics analysis of plasma samples from patients diagnosed with chronic kidney disease or kidney cancer (RCC), identifying dozens of phosphoproteins capable of distinguishing disease states from healthy controls. The study demonstrates the potential feasibility of our robust analytical pipeline for cancer signaling monitoring by tracking plasma EV phosphorylation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Renal Cell Carcinoma

SUBMITTER: Anton Iliuk

LAB HEAD: Andy Tao

PROVIDER: PXD017994 | Pride | 2020-06-15

REPOSITORIES: Pride

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Publications

Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification.

Iliuk Anton A Wu Xiaofeng X Li Li L Sun Jie J Hadisurya Marco M Boris Ronald S RS Tao W Andy WA

Journal of proteome research 20200528 7

The invasive nature and the pain caused to patients inhibit the routine use of tissue biopsy-based procedures for cancer diagnosis and surveillance. The analysis of extracellular vesicles (EVs) from biofluids has recently gained significant traction in the liquid biopsy field. EVs offer an essential "snapshot" of their precursor cells in real time and contain an information-rich collection of nucleic acids, proteins, lipids, and so on. The analysis of protein phosphorylation, as a direct marker ...[more]

PMID: 32396726

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

Dataset Information

Plasma-derived EV Phosphoproteomics through Chemical Affinity Purification

Publications

Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification.

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