biostudies-literatureZhu JNational Cancer InstituteNCI NIH HHS4901-4911https://www.ebi.ac.uk/biostudies/studies/S-EPMC8496948biostudies-literatureUnknown20(10)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 moleJournal of proteome researchColumn-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles.PMC8496948R50 CA221808R21CA189775R01 CA160254R21 CA189775Zhu JJi XZhang JTan ZLubman DMfalseColumn-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 mole2021-01-01T00:00:00Z2021 Oct2026-06-01T02:23:02.104Z2025-04-04T10:27:46.342ZS-EPMC84969483447350510.1021/acs.jproteome.1c00549