| Autor: |
Liu YK; Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, United States., Wu X; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States., Hadisurya M; Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, United States., Li L; Tymora Analytical Operations, West Lafayette, Indiana 47906, United States., Kaimakliotis H; Indiana University School of Medicine, Indianapolis, Indiana 46202, United States., Iliuk A; Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, United States.; Tymora Analytical Operations, West Lafayette, Indiana 47906, United States., Tao WA; Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, United States.; Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States.; Tymora Analytical Operations, West Lafayette, Indiana 47906, United States.; Purdue Institute for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States. |
| Abstrakt: |
Extracellular vesicle (EV) proteomics emerges as an effective tool for discovering potential biomarkers for disease diagnosis, monitoring, and therapeutics. However, the current workflow of mass spectrometry-based EV proteome analysis is not fully compatible in a clinical setting due to inefficient EV isolation methods and a tedious sample preparation process. To streamline and improve the efficiency of EV proteome analysis, here we introduce a one-pot analytical pipeline integrating a robust EV isolation approach, EV total recovery and purification (EVtrap), with in situ protein sample preparation, to detect urinary EV proteome. By incorporating solvent-driven protein capture and fast on-bead digestion, the one-pot pipeline enabled the whole EV proteome analysis to be completed within one day. In comparison with the existing workflow, the one-pot pipeline was able to obtain better peptide yield and identify the equivalent number of unique EV proteins from 1 mL of urine. Finally, we applied the one-pot pipeline to profile proteomes in urinary EVs of bladder cancer patients. A total of 2774 unique proteins were identified in 53 urine samples using a 15 min gradient library-free data-independent acquisition method. Taken altogether, our novel one-pot analytical pipeline demonstrated its potential for routine and robust EV proteomics in biomedical applications. |
