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PloS one2025; 20(1); e0315743; doi: 10.1371/journal.pone.0315743

Respiratory extracellular vesicle isolation optimization through proteomic profiling of equine samples and identification of candidates for cell-of-origin studies.

Abstract: Growing evidence supports the importance of extracellular vesicle (EV) as mediators of communication in pathological processes, including those underlying respiratory disease. However, establishing methods for isolating and characterizing EVs remains challenging, particularly for respiratory samples. This study set out to address this challenge by comparing different EV isolation methods and evaluating their impacts on EV yield, markers of purity, and proteomic signatures, utilizing equine/horse bronchoalveolar lavage samples. Horses can serve as effective translational animal models for respiratory studies due to similarities with human immune responses, shared environmental exposures, and naturally occurring respiratory diseases including asthma. Further, horses are long-lived large animals that allow for longitudinal sample collection, and provide large sample volume and cell yield, which are particularly useful since EV research is commonly limited by low sample yields. Here, EVs were isolated from horse bronchoalveolar lavage fluid (BALF) using four different methods (ultracentrifugation, microcentrifugation, and two sizes of size exclusion chromatography columns) and characterized by measuring particle counts, EV purity, total protein yield, and proteomic cargo, with a specific focus on vesicle surface marker expression potentially informing cell type of origin. We found that size exclusion chromatography yielded the highest particle counts, greatest EV purity markers and elevated vesicle surface marker expression. Overall proteomic profiles differed across isolation methods, with size exclusion chromatography clustering separately from centrifugation. Taken together, our results demonstrate that different isolation methods impact characteristics of EVs, notably that size exclusion chromatography, compared to centrifugation methods, resulted in higher EV purity and better characterized proteomic diversity, including information on EV cell-of-origin. This is the first study to characterize proteomic profiles of EVs following different isolation methods using equine BALF. The results of this study will pave the way for future studies using equine and human samples to characterize respiratory tract EVs.
Copyright: © 2025 Hickman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Publication Date: 2025-01-24 PubMed ID: 39854355PubMed Central: PMC11760557DOI: 10.1371/journal.pone.0315743Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research aimed to optimize the isolation of extracellular vesicles (EVs) from respiratory samples, specifically equine bronchoalveolar lavage fluid (BALF), by comparing different isolation methods and analyzing their effects on EV purity, yield, and proteomic profiles.
  • The study identified size exclusion chromatography as a superior method for obtaining highly pure EVs with better proteomic characterization, which can inform the cell of origin and facilitate respiratory disease research.

Background and Importance

  • Extracellular vesicles (EVs) are small membrane-bound entities released by cells that mediate communication between cells.
  • EVs play crucial roles in pathological processes, including respiratory diseases, making them important targets for study.
  • Isolating and characterizing EVs from respiratory samples is challenging due to sample complexity and low yield.
  • Horses are valuable animal models for respiratory studies because:
    • They share immunological and environmental similarities with humans.
    • They naturally develop respiratory diseases like asthma.
    • They provide large sample volumes and allow longitudinal sampling.

Objectives

  • To compare four different EV isolation methods from equine bronchoalveolar lavage fluid (BALF):
    • Ultracentrifugation
    • Microcentrifugation
    • Two sizes of size exclusion chromatography (SEC) columns
  • To evaluate EV yield, purity markers, total protein content, and the proteomic cargo associated with each isolation method.
  • To assess EV surface markers that could help identify the cell types from which the vesicles originated.

Methodology

  • Samples: Bronchoalveolar lavage fluid obtained from horses.
  • EV Isolation Techniques:
    • Ultracentrifugation: High-speed centrifugation to pellet EVs.
    • Microcentrifugation: Lower speed centrifugation targeting smaller vesicle populations.
    • Size Exclusion Chromatography (SEC): Two column types differing in pore size to separate EVs based on size.
  • Characterization:
    • Particle counts to estimate EV yield.
    • Measurement of EV purity to assess contamination by non-vesicular proteins.
    • Total protein quantification in isolated EVs.
    • Proteomic profiling using mass spectrometry to determine protein cargo.
    • Analysis of vesicle surface markers to infer cell-of-origin signatures.

Key Findings

  • Size exclusion chromatography (SEC) resulted in:
    • The highest particle counts, indicating better EV yield.
    • Greater EV purity as compared to centrifugation methods.
    • Elevated expression of vesicle surface protein markers, improving cell-of-origin informativeness.
  • Proteomic profiles varied depending on the isolation method:
    • SEC-isolated EVs clustered separately from those isolated via centrifugation methods, showing distinct protein cargo.
    • This indicates that isolation technique influences the molecular fingerprint of EVs.
  • Ultracentrifugation and microcentrifugation yielded EVs with less purity and lower representation of signature surface proteins.

Implications and Contributions

  • This study is the first to perform a comparative proteomic analysis of EVs isolated from equine BALF using multiple isolation methods.
  • SEC is demonstrated as a superior approach for isolating respiratory EVs with high purity and informative proteomic content.
  • The identification of vesicle surface markers represents a step forward in tracing EVs back to their cellular origins in the respiratory tract.
  • Findings facilitate improved EV research in equine models, which can be translated to human respiratory disease studies.
  • Better EV isolation and characterization techniques can advance understanding of EV roles in respiratory pathology and support biomarker discovery.

Future Directions

  • Apply optimized SEC isolation protocols in larger equine cohorts and diverse respiratory disease models.
  • Use identified cell-of-origin markers to map EV contribution in various pathological states.
  • Translate findings and protocols to human respiratory samples for clinical biomarker development.
  • Explore longitudinal EV proteomic changes to understand disease progression and therapeutic responses.

Cite This Article

APA
Hickman E, Carberry V, Carberry C, Cooper B, Mordant AL, Mills A, Sokolsky M, Herring LE, Alexis NE, Rebuli ME, Jaspers I, Sheats K, Rager JE. (2025). Respiratory extracellular vesicle isolation optimization through proteomic profiling of equine samples and identification of candidates for cell-of-origin studies. PLoS One, 20(1), e0315743. https://doi.org/10.1371/journal.pone.0315743

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 20
Issue: 1
Pages: e0315743
PII: e0315743

Researcher Affiliations

Hickman, Elise
  • Curriculum in Toxicology & Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
  • Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Carberry, Victoria
  • Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Carberry, Celeste
  • Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Cooper, Bethanie
  • Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina, United States of America.
Mordant, Angie L
  • UNC Michael Hooker Proteomics Core, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Mills, Allie
  • UNC Michael Hooker Proteomics Core, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Sokolsky, Marina
  • Center for Nanotechnology in Drug Delivery, UNC School of Medicine, Chapel Hill, North Carolina, United States of America.
Herring, Laura E
  • UNC Michael Hooker Proteomics Core, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
  • Department of Pharmacology, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
Alexis, Neil E
  • Curriculum in Toxicology & Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
  • Department of Pediatrics, UNC School of Medicine, Chapel Hill, North Carolina, United States of America.
Rebuli, Meghan E
  • Curriculum in Toxicology & Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
  • Department of Pediatrics, UNC School of Medicine, Chapel Hill, North Carolina, United States of America.
Jaspers, Ilona
  • Curriculum in Toxicology & Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
  • Department of Pediatrics, UNC School of Medicine, Chapel Hill, North Carolina, United States of America.
Sheats, Katie
  • Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina, United States of America.
Rager, Julia E
  • Curriculum in Toxicology & Environmental Medicine, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.
  • Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, UNC Chapel Hill, Chapel Hill, North Carolina, United States of America.

MeSH Terms

  • Animals
  • Horses
  • Extracellular Vesicles / metabolism
  • Bronchoalveolar Lavage Fluid / cytology
  • Bronchoalveolar Lavage Fluid / chemistry
  • Proteomics / methods
  • Proteome
  • Biomarkers / metabolism
  • Chromatography, Gel

Grant Funding

  • P30 ES025128 / NIEHS NIH HHS
  • P30 ES010126 / NIEHS NIH HHS
  • F32 ES036096 / NIEHS NIH HHS
  • R01 ES035878 / NIEHS NIH HHS
  • T32 ES007126 / NIEHS NIH HHS
  • P30 CA016086 / NCI NIH HHS

Conflict of Interest Statement

The authors have declared that no competing interests exist.

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Citations

This article has been cited 1 times.
  1. Pham TTH, Sakamoto H, Hasegawa T, Sakamoto C, Suye SI, Chuang HS. Small extracellular vesicle-associated surface protein biomarkers: emerging roles, opportunities, and challenges in diagnostics.. Front Bioeng Biotechnol 2025;13:1714972.
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