Precipitation of small extracellular blood vesicles under acidic conditions and the use of this method to isolate vesicles of neuronal origin

Relevance . Studies of the composition and properties of small extracellular vesicles (sEVs) are becoming more and more important due to their usefulness for the diagnosis and therapy of many pathologies. However, reliable universal methods of sEVs isolation and study have not been developed yet. Objective. To develop a modification of the method of sEVs isolation and to use the modified method for isolation of sEVs of neuronal origin. Materials and Methods . Using polyethylene glycol (PEG) precipitation at different pH and ionic strength values, sEVs were isolated from the blood of healthy volunteers. Immunoprecipitation was used to isolate sEVs of neuronal origin. Neuronal sEVs were biotinylated, and biotinylated proteins were identified by mass spectrometry. Results . As a result of this work, a modification of the PEG precipitation method at acidic pH values was developed, which allowed to increase the yield of neuronal sEVs several times. The surface proteins of sEVs were identified; these proteins were serum albumin, apolipoprotein B, complement system components C1r, C1q, C1s, C3 and immunoglobulin heavy chains. Conclusion . PEG precipitation under acidic conditions allows the isolation of more neuronal sEVs from serum than PEG precipitation did in neutral medium. At the same time, neuronal sEVs in blood most likely exist in complex with blood proteins, representing the so-called protein “corona”.