Automated Extracellular Vesicles Extractor Based on Nanotechnology for High Throughput Sample Preparation and Phosphoproteomics Analysis

Abstract

Extracellular vesicles (EVs) play important roles in the diagnosis and treatment of diseases because of its lavish bioinformatic molecular contents. However, how to separate plentiful of extracellular vesicles in a short time with high consistency is still a problem. Here, we’d like to introduce an automated extracellular vesicles capturing machine named EVenrich (Extracellular Vesicles Enrichment) that helps realize capturing EVs of biosamples in a high-throughput manner. Incorporating EVenrich with EVtraps-a kind of biological magnetic beads capturing extracellular vesicles specifically, we were able to isolate EVs from up to 48 urine samples in 60 min, with similar quality and quantity and superior stability to manual capture. In clinical urine EVs analysis, over 14000 peptide fragments and 2000 proteins were detected by automated capture, which was similar to manual capture and significantly superior to ultracentrifugation. We further collected urine samples from 21 patients with negative prostate biopsy results and 21 patients with confirmed prostate cancer. Every three samples were mixed into a group to make mixed samples of 7 control groups and 7 disease groups. After exosome extraction and exosome proteomics / phosphorylated proteomics analysis, we found 211 overexpressed proteins and 187 overexpressed phosphorylated peptides corresponding to 40 phosphorylated proteins as potential biomarkers for prostate cancer. In addition, qRT-PCR results showed that the ratio of miR-125b to miR-145 could well distinguish prostate cancer patients from non-prostate cancer patients, suggesting that it could be used as a potential marker of prostate cancer.