Phosphoproteomics Protocol for the Identification of Novel Biomarkers from Extracellular Vesicles of Cancerous Cells.

In this chapter, we describe a general sample preparation workflow for small extracellular vesicles (sEVs) from breast cancer cell lines using techniques such as protein solubilization, enzymatic digestion, and Fe3+-immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO2) enrichment prior to mass spectrometry-based proteomic analysis. This workflow can be used to perform global phosphoproteomics of sEVs enabling the study of tumor biology and the discovery of biomarkers for cancer diagnosis and treatment.