Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Gabriel Jakob Trauner; Dominika Bernath-Nagy; Melek S&#252;kran Kalinyaprak; Sabine Merker; Marcin Luzarowski; Florian Leuschner; Norbert Frey; Evangelos Giannitsis; Jona  Benjamin Krohn

doi:10.3791/67083

Aislamiento, caracterización y análisis proteómico de vesículas extracelulares derivadas del plas...

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JoVE Journal Medicine

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Aislamiento, caracterización y análisis proteómico de vesículas extracelulares derivadas del plasma para el descubrimiento de biomarcadores cardiovasculares

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05:30 min

January 31, 2025

DOI: 10.3791/67083-v

Gabriel Jakob Trauner*¹, Dominika Bernath-Nagy*^1,2, Melek Sükran Kalinyaprak¹, Sabine Merker³, Marcin Luzarowski³, Florian Leuschner^1,4, Norbert Frey^1,4, Evangelos Giannitsis¹, Jona Benjamin Krohn^1,4

¹Department of Cardiology, Angiology and Pulmonology,University Hospital Heidelberg, ²Heart and Vascular Center,Semmelweis University, ³Core Facility for Mass Spectrometry and Proteomics,Centre for Molecular Biology at Heidelberg University (ZMBH), ⁴German Centre for Cardiovascular Research (DZHK), Partner site Heidelberg/Mannheim

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Overview

This article presents a protocol for the isolation and characterization of human plasma-derived extracellular vesicles (EVs). It emphasizes the importance of maintaining EV integrity for accurate biomarker discovery in clinical settings.

Key Study Components

Area of Science

Biomarker research
Extracellular vesicles
Proteomics

Background

Extracellular vesicles are emerging as significant biomarkers in various diseases.
They contain proteins and RNA that can provide insights into disease states.
Challenges include the preservation of EVs and the dynamics of plasma acquisition.
A robust protocol is essential for reproducible results in biomarker analysis.

Purpose of Study

To provide a hands-on methodology for EV biomarker discovery.
To facilitate the implementation of this protocol in emergency department settings.
To enhance the understanding of EV molecular cargo in cardiovascular diseases.

Methods Used

Isolation of human plasma-derived extracellular vesicles.
Characterization and quantification of EVs.
Label-free analysis of the EV proteome.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomic analysis.

Main Results

Developed a stringent protocol to prevent EV degradation.
Demonstrated the potential of EVs as biomarkers for cardiovascular diseases.
Provided a workflow for effective proteomic analysis of EVs.
Highlighted the importance of EV preservation in clinical applications.

Conclusions

The methodology enhances the reliability of EV biomarker discovery.
It supports the integration of EV analysis into routine clinical practice.
Future research can build on this protocol to explore EVs in other disease contexts.

Frequently Asked Questions

What are extracellular vesicles?

Extracellular vesicles are small membrane-bound particles released by cells that carry molecular information, including proteins and RNA.

Why are EVs important in biomarker research?

EVs contain molecular cargo that can provide insights into disease states, making them valuable for biomarker discovery.

What challenges exist in EV biomarker discovery?

Major challenges include the preservation of EVs during collection and the dynamics of their release from cells.

How does the protocol ensure EV integrity?

The protocol includes stringent steps to minimize degradation and maintain the stability of EVs during processing.

What techniques are used for analyzing EVs?

The study employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for detailed proteomic analysis of EVs.

Este artículo describe una metodología para el aislamiento, caracterización y cuantificación de vesículas extracelulares (EV) derivadas del plasma humano y presenta un flujo de trabajo para el análisis sin etiquetas del proteoma EV utilizando cromatografía líquida-espectrometría de masas en tándem (LC-MS/MS).

Las vesículas extracelulares circulantes como biomarcadores son un campo relativamente nuevo en la investigación de biomarcadores con un inmenso potencial. Nuestro protocolo tiene como objetivo proporcionar un enfoque práctico para implementar el descubrimiento y la detección de biomarcadores unidos a vesículas extracelulares en un entorno de servicio de emergencias. La evidencia emergente ha demostrado que la carga molecular que se encuentra en las vesículas extracelulares y la circulación contiene proteínas y ARN de interés en múltiples contextos de enfermedad.

Varios nuevos biomarcadores de vesículas extracelulares encerrados se han relacionado con el diagnóstico y el pronóstico de diversas entidades de enfermedad cardiovascular. Los principales desafíos en el campo del descubrimiento de biomarcadores de vesículas extracelulares circulantes son la preservación de las vesículas extracelulares y la adquisición de plasma que depende de la dinámica de liberación de EV. Por lo tanto, es necesario un protocolo estricto integrado en la rutina clínica para evitar la degradación de EV y mantener resultados reproducibles, particularmente cuando se aplican métodos de análisis a gran escala como la proteómica.

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