Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles Through Automated and High-Throughput Isolation

Overview

Journal Cells

Publisher MDPI

Specialties Biochemistry
Biophysics
Cell Biology
Molecular Biology

Date 2022 Jul 9

PMID 35805153

Authors

Hao Zhang

Yu-Han Cai

Yajie Ding

Guiyuan Zhang

Yufeng Liu

Jie Sun

Yuchen Yang

Zhen Zhan

Anton Iliuk

Zhongze Gu

Yanhong Gu

W Andy Tao

Affiliations

Soon will be listed here.

Abstract

Extracellular vesicles (EVs) play an important role in the diagnosis and treatment of diseases because of their rich molecular contents involved in intercellular communication, regulation, and other functions. With increasing efforts to move the field of EVs to clinical applications, the lack of a practical EV isolation method from circulating biofluids with high throughput and good reproducibility has become one of the biggest barriers. Here, we introduce a magnetic bead-based EV enrichment approach (EVrich) for automated and high-throughput processing of urine samples. Parallel enrichments can be performed in 96-well plates for downstream cargo analysis, including EV characterization, miRNA, proteomics, and phosphoproteomics analysis. We applied the instrument to a cohort of clinical urine samples to achieve reproducible identification of an average of 17,000 unique EV peptides and an average of 2800 EV proteins in each 1 mL urine sample. Quantitative phosphoproteomics revealed 186 unique phosphopeptides corresponding to 48 proteins that were significantly elevated in prostate cancer patients. Among them, multiple phosphoproteins were previously reported to associate with prostate cancer. Together, EVrich represents a universal, scalable, and simple platform for EV isolation, enabling downstream EV cargo analyses for a broad range of research and clinical applications.

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