Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation

Abstract

Abstract Background: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Results: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: i) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, ii) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and iii) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, which allows subsequent characterization and functional analysis of EVs. We optimized all steps of the protocol to enhance peptide exposure on the beads leading to a final procedure that incubates 0.5mg of peptide for 10 minutes with 10µl of Long-arm Cobalt agarose before and overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be performed on the agarose beads by simple adding lysis or nucleic-acid extraction buffers. Alternatively, EVs can be gently eluted by competition with imidazole, rendering a fully competent EV preparation. Conclusions: This new isolation methodology is based on the recognition of general membrane characteristics of EVs and thus can be a good option for a total isolation of EVs without a bias based on the surface markers. It can be used in any species EV sample, enabling this approach to samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, and less time-consuming than traditional methods, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down according to operator needs, with the possibility of analyzing very low amounts of sample. Finally, it is compatible with any downstream analyses thanks to the gentle elution procedure.