Details

Autor(en) / Beteiligte

• Pham, Quang Nghia
• Winter, Marnie
• Milanova, Valentina
• Young, Clifford
• Condina, Mark R
• Hoffmann, Peter
• Pham, Nguyen T. H
• Tung, Tran Thanh
• Losic, Dusan
• Thierry, Benjamin

Titel

Magnetic enrichment of immuno-specific extracellular vesicles for mass spectrometry using biofilm-derived iron oxide nanowires

Ist Teil von

• Nanoscale, 2023-01, Vol.15 (3), p.1236-1247

Ort / Verlag

England: Royal Society of Chemistry

Erscheinungsjahr

2023

Quelle

MEDLINE

Beschreibungen/Notizen

• 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 placenta-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 ± 0.7 × 10 9 and 7.1 ± 1.2 × 10 9 , 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 immuno-specific EV enrichment for LC-MS/MS method provides a low-cost and highly-scalable yet efficient, high-throughput approach for quality EV proteomic studies. Magnetic extracellular vesicle (EV) enrichment using antibody conjugated bacteria-derived iron oxide nanowires coupled with mass spectrometry-based proteome profiling enables efficient EV subtype enrichment and reproducible proteomics.