Column-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles

Serum-derived
extracellular vesicles (EVs) are a promising source
of biomarkers; however, major challenges in EV separation and proteomic
profiling remain for isolating EVs from a small amount, that is, on
the microliter scale, of human serum while minimizing the contamination
of blood proteins and lipoprotein particles coeluting in EV preparations.
Herein we have developed a column-based CD9-antibody-immobilized high-performance
liquid chromatography immunoaffinity chromatography­(CD9-HPLC-IAC)
technology for EV isolation from a microliter scale of serum for downstream
proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation
from 40 μL of serum in 30 min with a yield of 8.0 × 109 EVs, where EVs were further processed with a postcolumn cleaning
step using the 50 kDa molecular weight cut-off filter for the buffer
exchange, concentration, and reduction of potentially coeluting serum
proteins. In total, 482 proteins were identified in EVs by using liquid
chromatography tandem mass spectrometry, including the common exosomal
markers such as CD63, CD81, CD82, Alix, and TSG101. The statistical
analysis of EV protein content showed that the top 10 serum proteins
in EVs were significantly decreased by using the CD9-HPLC-IAC method
compared with the use of ultracentrifugation (p =
0.001) and size exclusion chromatography (p = 0.009),
and apolipoproteins were significantly reduced 4.8-fold compared with
the SEC method (p < 0.001). The result demonstrates
the potential of the CD9-HPLC-IAC method for the efficient isolation
and proteomic characterization of EVs from a microscale volume of
serum.