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

Summary

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Abstract

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

Introduction

Extracellular vesicles (EV), by nomenclature not uniformly defined, are nanoparticles surrounded by a lipid bilayer and released by various cell types, lacking the ability to replicate¹. This diverse group includes exosomes, a subpopulation of EV of endosomal origin, typically ranging from approximately 40 nm to 160 nm in diameter². Detectable in numerous body fluids³, EVs facilitate intercellular communication by transferring various active biomolecules such as proteins, mRNA, microRNA, and lipids. Thus, EV provide information about their cell of origin through cell-specific surface markers and b....

Protocol

For this protocol, an exemplary patient cohort was recruited comprising three healthy control patients with no signs of apparent or underlying cardiovascular disease and two patients with non-ST elevated myocardial infarction (NSTEMI). Prior approval was granted by the Institutional Review Board of the Medical Faculty of the University of Heidelberg (IRB approval #S-351/2015), and written consent was obtained from all patients prior to recruitment. Patients were recruited from the Chest Pain Unit of the Department of Cardiology at University Hospital Heidelberg based on initial clinical presentation, medical history, and the results of diagnostic testing.

Results

EV were isolated from plasma samples (n = 3) of patients without overt cardiovascular disease, as well as from patients with non-ST elevation myocardial infarction (NSTEMI; n = 2), using the established differential ultracentrifugation protocol. Adequate separation of plasma EV was confirmed by immunoblotting of EV-enriched proteins TSG-101, annexin 5 (Anx5), and CD9 in EV isolates compared to EV-depleted plasma from the same patients (Figure 1A). Transferrin, serving as a negative control, .......

Discussion

This protocol provides a real-world, step-by-step, ready-to-use methodology for the separation and characterization of plasma EV, as well as an introduction to an unlabeled proteome analysis suitable for integration into routine clinical practice. A detailed description and strict adherence to a unified methodology for EV isolation from plasma samples are important to ensure reproducibility of obtained results. Current literature indicates that pre-analytical conditions can significantly impact subsequent measurements an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4x Laemmli sample bufffer	Bio-rad	1610747
10x RIPA-Buffer	Abcam	ab156034
26.3 mL Polycarbonate Bottle with Cap Assembly for Ultracentrifugation	Beckman Coulter	355654
5810R Benchtop centrifuge	Eppendorf	5811000015
Acetonitrile (ACN)	Biosolve	0001204101BS
Dithiothreitol (DTT)	Sigma-Aldrich	43816-10ML
Dulbecco's Phosphate buffered saline (PBS)	Sigma-Aldrich	D8537
Formic Acid 99% ULC/MS 100	Biosolve	0006914143BS
Histopaque - 1077	Sigma-Aldrich	10771
Iodoacetamide (IAA)	Sigma-Aldrich	I6125-5G
Mass Spectrometer Orbitrap Q Exactive HF	Thermo Fisher Scientific	IQLAAEGAAPFALGMBDK
MOPS SDS running buffer	Thermo Fisher	B0001
NanoSight NS300	Malvern Panalytical	NS300
Nanosight NTA 3.2 Software	Malvern Panalytical
NuPAGE 4 bis 12 %, Bis-Tris protein gels	Invitrogen	NP0323
Optima XPN-80 floor-standing ultracentrifuge	Beckman Coulter	521-4180
PageRuler Plus Prestained Protein Ladder	Thermo Fisher Scientific	26619
Protease/Phosphatase Inhibitor Cocktail (100X)	Cell Signaling Technology	5872S
Protein marker	Thermo Fisher	26616
Proteome Discoverer 2.5	Thermo Fisher
Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer	Thermo Fisher Scientific	IQLAAEGAAPFALGMBDK
Quick stain coomasssie	Serva	35081.01
ReproSil-Pur 120 C18-AQ, 1.9 µm 1 g	Dr. Maisch	r119.aq.0001
SDS-PAGE commercial gel	Thermo Fisher	NW00100BOX
S-Monovette Citrat 9NC 0.106 mol/l 3,2%	Sarstedt	02.1067.001
Speed vac concentrator	Savant
Swiss-Prot (Uniprot) Homo sapiens (UP000005640, June 2020) protein database	UniProt	https://www.uniprot.org/proteomes/UP000005640
Triethylammonium bicarbonate buffer (TEAB)	Sigma-Aldrich	T7408
Trifluoroacetic acid (TFA) for HPLC >99%, 100 mL	Sigma-Aldrich	302031-100ML
Trypsin MS-Grade	Thermo Fisher	90058
Type 50.2 Ti Fixed-Angle Rotor	Beckman Coulter	337901
UHPLC Dionex Ultimate 3000	Thermo Fisher Scientific	ULTIM3000RSLCNANO
Water	Biosolve	0023214102BS