Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles Journal Article


Authors: Wan, Z.; Gu, J.; Balaji, U.; Bojmar, L.; Molina, H.; Heissel, S.; Pagano, A. E.; Peralta, C.; Shaashua, L.; Ismailgeci, D.; Narozniak, H. K.; Song, Y.; Jarnagin, W. R.; Kelsen, D. P.; Bromberg, J.; Pascual, V.; Zhang, H.
Article Title: Optimization of ultracentrifugation-based method to enhance the purity and proteomic profiling depth of plasma-derived extracellular vesicles and particles
Abstract: Circulating extracellular vesicles and particles (EVPs) are being investigated as potential biomarkers for early cancer detection, prognosis, and disease monitoring. However, the suboptimal purity of EVPs isolated from peripheral blood plasma has posed a challenge of in-depth analysis of the EVP proteome. Here, we compared the effectiveness of different methods for isolating EVPs from healthy donor plasma, including ultracentrifugation (UC)-based protocols, phosphatidylserine-Tim4 interaction-based affinity capture (referred to as “PS”), and several commercial kits. Modified UC methods with an additional UC washing or size exclusion chromatography step substantially improved EVP purity and enabled the detection of additional proteins via proteomic mass spectrometry, including many plasma membrane and cytoplasmic proteins involved in vesicular regulation pathways. This improved performance was reproduced in cancer patient plasma specimens, resulting in the identification of a greater number of differentially expressed EVP proteins, thus expanding the range of potential biomarker candidates. However, PS and other commercial kits did not outperform UC-based methods in improving plasma EVP purity. PS yielded abundant contaminating proteins and a biased enrichment for specific EVP subsets, thus unsuitable for proteomic profiling of plasma EVPs. Therefore, we have optimized UC-based protocols for circulating EVP isolation, which enable further in-depth proteomic analysis for biomarker discovery. © 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.
Keywords: biomarkers; proteomics; early cancer detection; extracellular vesicles and particles (evps)
Journal Title: Journal of Extracellular Biology
Volume: 3
Issue: 7
ISSN: 2768-2811
Publisher: Wiley Blackwell  
Date Published: 2024-07-01
Start Page: e167
Language: English
DOI: 10.1002/jex2.167
PROVIDER: scopus
PMCID: PMC11263976
PUBMED: 39045341
DOI/URL:
Notes: Source: Scopus
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  1. Jacqueline Bromberg
    141 Bromberg
  2. William R Jarnagin
    903 Jarnagin
  3. David P Kelsen
    537 Kelsen
  4. Yi Song
    10 Song