Mag-Net Strong Anion Exchange Enables Isolation of Ovarian Cancer Ascites Extracellular Vesicles for Proteomic Biomarker Discovery.

Extracellular vesicles (EVs) are nanoscale particles secreted by all cells and present in all biological fluids, where they carry molecular cargo reflective of health and disease states. Their diagnostic potential is often obscured by the high abundance of non-EV proteins and lipoproteins (e.g., albumin, apolipoproteins) that complicate proteomic analysis of primary biofluids, such as ascites fluid. Conventional isolation strategies face a persistent trade-off between EV purity and yield. To overcome this, a magnetic bead-based protocol (Mag-Net) to enrich EVs according to electrochemical surface charge using strong anion-exchange chemistry (SAX) was adapted for proteomics. Our workflow is specifically adapted to ascites fluid from human or murine sources. This approach effectively separates EVs from high-abundance proteins and lipoproteins, enabling proteomic profiling from as little as 2 μL of ascites fluid. Demonstrated in both murine and human ovarian cancer models, Mag-Net offers a reproducible, scalable, and automation-ready solution for EV isolation from various biofluids. Key features • Extracellular vesicles (EVs) from murine and human ascites fluid are effectively enriched using Mag-Net beads. • EVs are effectively captured and eluted from Mag-Net beads to support Raman spectroscopy, nanoparticle tracking analysis, and atomic force microscopy. • EV isolation by Mag-Net provides robust proteomic depth obtained by mass spectrometry. • Robust proteomic data can be obtained from input volumes ranging from 2 to 100 μL of ascites.