Proteomics, Phosphoproteomics and Mirna Analysis of Circulating Extracellular Vesicles through Automated and High-Throughput Isolation
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
Hao Zhang
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Yu-Han Cai
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Yajie Ding
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Guiyuan Zhang
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Yufeng Liu
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Jie Sun
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Yuchen Yang
Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing 210029, China
Zhen Zhan
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Anton Iliuk
Tymora Analytical Operations, West Lafayette, IN 47906, USA
Zhongze Gu
State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China
Yanhong Gu
Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing 210029, China
W. Andy Tao
Tymora Analytical Operations, West Lafayette, IN 47906, USA
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.
