One-Pot Analytical Pipeline for Efficient and Sensitive Proteomic Analysis of Extracellular Vesicles

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.