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Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification
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Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis.

Laura Botelho Merij1, Luana Rocha da Silva2, Lohanna Palhinha3

  • 1Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; Programa de Pós-Graduação em Ciências Biológicas, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil.

Journal of Thrombosis and Haemostasis : JTH
|January 26, 2024
PubMed
Summary

Lipoprotein contamination in blood plasma extracellular vesicles (EVs) was reduced using gradient ultracentrifugation (G-UC) and size-exclusion chromatography (SEC). This improved EV purity for enhanced analysis of thromboinflammation and biomarker discovery.

Keywords:
EV isolationEV proteomicsblood plasmaextracellular vesicleslipoproteins

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Proteomics

Background:

  • Blood plasma extracellular vesicles (EVs) are crucial for biomarker discovery and understanding inflammation and thrombosis.
  • Lipoprotein contamination is a significant challenge in plasma EV isolation, potentially biasing results.
  • Lipoproteins influence cell signaling, metabolism, and thromboinflammation, necessitating their removal for accurate EV analysis.

Purpose of the Study:

  • To develop a method for isolating plasma EVs with reduced lipoprotein contamination.
  • To improve the purity of EV samples for more reliable thromboinflammatory analyses.

Main Methods:

  • Density-based gradient ultracentrifugation (G-UC) was employed for initial lipoprotein depletion.
  • Extracellular vesicles (EVs) were subsequently isolated using size-exclusion chromatography (SEC) or serial centrifugation (SC).
  • Isolated EVs were characterized by size, concentration, cellular source, ultrastructure, and proteomic analysis.

Main Results:

  • G-UC effectively separated lipoproteins, enabling cleaner EV isolation via SEC or SC.
  • Proteomic, cholesterol, and apoB-100 analyses confirmed significant lipoprotein reduction in isolated EVs.
  • Lipoprotein depletion did not alter EV cellular source, gene ontology, or ultrastructure, but enhanced the detection of low-abundance proteins and improved in vitro thromboinflammatory responses.

Conclusions:

  • The combination of G-UC and SEC effectively reduces lipoprotein contamination in plasma EVs.
  • This method yields highly pure EVs without compromising their fundamental characteristics.
  • The improved purity has significant implications for functional assays, proteomics, and lipidomics of EVs.