Magnetic Enrichment of Immuno-Specific Extracellular Vesicles for Mass Spectrometry Using Biofilm-Derived Iron Oxide Nanowires

Abstract

Immuno-specific enrichment of extracellular vesicles (EVs) can provide important information into cellular pathways underpinning various pathologies and for non-invasive diagnostics, including mass spectrometry-based analyses. Herein, we report an optimised protocol for immuno-magnetic enrichment of specific EV subtypes and their subsequent processing with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, we conjugated placental alkaline phosphatase (PLAP) antibodies to magnetic iron oxide nanowires (NWs) derived from bacterial biofilms and demonstrated the utility of this approach by enriching placental specific EVs (containing PLAP) from cell culture media. We demonstrate efficient PLAP+ve EV enrichment for both NW-PLAP and Dynabeads-PLAP, with high PLAP protein recovery (83.7{+/-}8.9% and 83.2{+/-}5.9%, respectively), high particle-to-protein ratio (7.5{+/-}0.7x109 and 7.1{+/-}1.2x109, respectively), and low non-specific binding of non-target EVs (7{+/-}3.2% and 5.4{+/-}2.2%, respectively). Furthermore, our optimized EV enrichment and processing approach identified 2518 and 2545 protein groups with LC-MS/MS for NW-PLAP and Dynabead-PLAP, respectively, with excellent reproducibility (Pearson correlation 0.986 and 0.988). These findings demonstrate that naturally occurring iron oxide NWs have comparable performance to current gold standard immune-magnetic beads. The optimized immunospecific EV enrichment for LC-MS/MS method provides a low-cost and highly-scalable yet efficient, high-throughput approach for quality EV proteomic studies.