Open AccessColumn-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles
Author(s) -
Jianhui Zhu,
Jie Zhang,
Xiaohui Ji,
Zhijing Tan,
David M. Lubman
Publication year - 2021
Publication title -
journal of proteome research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.644
H-Index - 161
eISSN - 1535-3907
pISSN - 1535-3893
DOI - 10.1021/acs.jproteome.1c00549
Subject(s) - chromatography , chemistry , high performance liquid chromatography , affinity chromatography , size exclusion chromatography , blood proteins , tandem mass spectrometry , extracellular vesicles , microscale thermophoresis , mass spectrometry , biochemistry , biology , enzyme , microbiology and biotechnology
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 × 10 9 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.