A simple capillary isoelectric focusing method as the novel strategy for the isoelectric point measurement of exosomes and its application in disease diagnosis.

A straightforward capillary isoelectric focusing (cIEF) method is established with a isoelectric point (pI) range spanning from 3.5 to 7.0, exhibiting excellent linearity and repeatability, with an R value of 0.9937 and migration time RSDs for all standard proteins below 0.3%. Subsequently, this method was applied to model exosomes derived from cell lines and healthy human serum, and the peak attributions of exosomes were identified using DiI labeled exosomes and lysed exosomes. The reproducibility of this method in exosome detection was also validated, as the RSDs of all pI values were less than 1%. Then, we observed a significant increase in the pI of exosomes with higher cholesterol content, irrespective of whether they originated from cell culture medium or mouse plasma. Notably, serum exosomes from healthy volunteers exhibited higher pI values compared to those from hepatocellular carcinoma patients, suggesting a potential diagnostic perspective for cancer. These findings underscore the significance of pI measurement in reflecting modifications in exosomal lipid membrane composition and their implications in biological functions mediated by exosomes.