Developing an Optimized Workflow for Exosome Proteomic Analysis

This capstone project aimed to develop a reproducible, high-yield, and automatable workflow for the isolation and proteomic analysis of exosomal proteins from human plasma. Traditional methods like ultracentrifugation and precipitation are limited by poor reproducibility, low yield, and contamination with non-exosomal proteins. To address these issues, a bead-based immunoaffinity approach using Inoviq’s EXO-NET technology was optimized and automated on the Agilent Bravo liquid handling system. Proteolytic digestion was refined using magnetic MPSP beads to improve peptide recovery and mass spectrometry compatibility.

The optimized workflow demonstrated superior performance in protein yield, exosome marker enrichment, and data reproducibility compared to five commercial kits and traditional isolation methods. LC-MS/MS analysis identified ~94 of the top 100 ExoCarta proteins, confirming high sensitivity and depth of coverage. Plasma samples, particularly EDTA-treated, outperformed serum in consistency and protein diversity. This workflow offers a scalable and reliable platform for exosome-based biomarker discovery and is well-suited for clinical and research applications.