Plasma-Derived Extracellular Vesicle Phosphoproteomics through Chemical Affinity Purification

Area of Science:

• Biochemistry
• Proteomics
• Oncology

Background:

• Tissue biopsies for cancer diagnosis are invasive and painful.
• Liquid biopsies using extracellular vesicles (EVs) offer a less invasive alternative.
• EVs contain proteins and nucleic acids reflecting cellular states, making them valuable biomarkers.

Purpose of the Study:

• To develop a rapid and efficient method for extracellular vesicle (EV) isolation from human plasma for phosphoproteomics analysis.
• To establish a robust analytical pipeline for EV phosphoproteomics to aid in cancer diagnosis and surveillance.
• To identify EV phosphoproteins that can distinguish between healthy individuals and patients with chronic kidney disease or kidney cancer.

Main Methods:

• Developed EVtrap (extracellular vesicle total recovery and purification), a chemical affinity-based method for rapid EV isolation.
• Utilized high-performance mass spectrometry (MS) for EV phosphoproteomics analysis of plasma samples.
• Quantitatively analyzed EV phosphoproteomics data from patients with kidney disease/cancer and healthy controls.

Main Results:

• EVtrap isolated over 16,000 unique peptides (2238 proteins) from 5 μL of plasma, exceeding ultracentrifugation recovery.
• Identified over 5500 unique phosphopeptides (approx. 1600 phosphoproteins) from 1 mL of plasma.
• Discovered dozens of phosphoproteins capable of differentiating disease states (kidney disease/cancer) from healthy controls.

Conclusions:

• The EVtrap method combined with MS provides a robust pipeline for plasma EV phosphoproteomics.
• This approach enables sensitive detection and quantification of EV phosphoproteins for disease monitoring.
• Plasma EV phosphoproteomics holds significant potential for non-invasive cancer signaling monitoring and early disease detection.