Abstract
Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs. By using a partially membrane-permeable disuccinimidyl suberate crosslinker, proteins on the EV outer-surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane-enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high-resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.
| Original language | English |
|---|---|
| Article number | e12245 |
| Pages (from-to) | 1-15 |
| Journal | Journal of Extracellular Vesicles |
| Volume | 11 |
| Issue number | 8 |
| DOIs | |
| Publication status | Published - Aug 2022 |
Bibliographical note
Funding Information:We thank Martijn C. Koorengevel (Membrane Biochemistry and Biophysics group, Utrecht University) for support and training in the use of ultracentrifuges, and the Laboratory of Experimental Cardiology (UMC Utrecht) for access to NTA equipment. We thank Dr. Joost Snijder (Utrecht University) for the assistance in NS‐TEM measurements. We acknowledge support for this research through the NWO funded National Road Map for Large‐scale Infrastructures program X‐Omics (Project 184.034.019) embedded in the Netherlands Proteomics Centre.
Funding Information:
We thank Martijn C. Koorengevel (Membrane Biochemistry and Biophysics group, Utrecht University) for support and training in the use of ultracentrifuges, and the Laboratory of Experimental Cardiology (UMC Utrecht) for access to NTA equipment. We thank Dr. Joost Snijder (Utrecht University) for the assistance in NS-TEM measurements. We acknowledge support for this research through the NWO funded National Road Map for Large-scale Infrastructures program X-Omics (Project 184.034.019) embedded in the Netherlands Proteomics Centre.
Publisher Copyright:
© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.