Abstract
We previously reported a novel method for isolating extracellular vesicles (EVs) using a polyamine solution through liquid chromatography-tandem mass spectrometry analysis of the culture supernatant of adherent cells, NCI-N87, and demonstrated its efficacy from multiple perspectives. Herein, K562 cells, a suspension cell line, were used to investigate the differences between the adherent cells. Furthermore, we elucidated the mechanism of EV isolation by focusing on the proteins’ isoelectric point (pI) on the EV surface.
Samples were prepared using a conditioned medium from the NCI-N87 and K562 cell lines. The conditioned medium was analyzed after replacing the medium with a serum-free medium and culturing for 48 h. The polyamine precipitation (PA) method was compared with the ultracentrifugation (UC) method.
Using K562 cells, the recovery of EVs using the PA method allowed the detection of common EV marker proteins. The gene ontology analysis also indicated that EV-related terms ranked the highest, similar to the UC method. These results demonstrate that the PA method is also effective for recovering EVs from suspended cells, such as K562 cells.
When the average pI values were calculated with the abundance and the pI values for the surface proteins in the collected EVs, the values with the PA method were lower than those with the UC method. These results suggest that electrostatic interactions bring the easy recovery of EVs with polyamines.
