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 biomolecules, with their properties strongly influenced by the condition of the parent cell and its environment⁴. These characteristics have led to a growing interest in the potential role of EV as biomarkers, particularly in the context of cardiovascular diseases.

Numerous in vitro and in vivo studies have demonstrated that myocardial hypoxic stress leads to an increased release of EV^5,6,7. Contemporary research on EV cargo has largely focused on EV-bound microRNA, which has the potential to serve as a biomarker in the diagnosis of various cardiovascular diseases^8,9,10. In contrast, evidence on the circulating EV proteome remains relatively scarce, with even fewer studies investigating plasma EV in cardiovascular patients. In two comprehensive studies on plasma-derived EV from patients suffering myocardial infarction, the authors identified a specific ischemia-induced EV proteome profile with potential diagnostic relevance^6,11.

The methodological processing of EV has historically proven challenging, and currently, there is no definitive recommendation for the optimal approach to EV isolation, characterization, and quantification. Commonly used methods for EV isolation include differential ultracentrifugation, density-gradient centrifugation, and filtration methods such as size-exclusion chromatography¹. According to current consensus guidelines, the characterization of EV isolates should include evidence of at least three typical EV surface protein markers, such as tetraspanins or annexins, combined with an imaging modality¹. To examine EV cargo at the protein level, antibody-based methods such as Western blot or ELISA are most frequently utilized.

Given the methodological challenges associated with the isolation and processing of circulating EV, this protocol presents a comprehensive pathway from patient recruitment and sample collection to subsequent EV isolation, characterization, and quantification of plasma EV isolates. Additionally, this study showcases a workflow for the immediate isolation of plasma-derived EV from patients presenting to the emergency department (Chest Pain Unit) at a tertiary care center in southwestern Germany, followed by the label-free analysis of disease-specific plasma EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The aim is to facilitate a high-throughput analysis to identify differentially enriched EV-bound proteins across a large cohort of patients with diverse ischemic, congenital, or (auto-)immune cardiovascular diseases at the initial diagnosis and throughout disease progression and/or resolution. This proteomic screening approach seeks to identify patterns of EV-specific protein enrichment associated with distinct disease pathways, with the ultimate goal of uncovering novel EV-bound protein biomarkers to enhance current diagnostics and therapeutic monitoring in cardiovascular disease.

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.

Inclusion criteria for healthy control patients included atypical or nonspecific clinical presentation, cardiac biomarker levels within normal limits, no history of cardiovascular disease, and normal findings on any further diagnostic testing. Exclusion criteria included a history of malignancy or cytostatic therapy within the last five years, hemodialysis, familial hyperlipidemia syndromes, and any history of cardiovascular disease. NSTEMI was diagnosed following current guidelines¹², with hemodynamically relevant coronary stenosis confirmed by coronary angiography. For each patient, up to 36 mL of blood was collected from the antecubital vein into citrate-supplemented collection tubes via standard phlebotomy¹³, and blood samples were immediately transported at 4 °C for further processing. Details of the reagents and equipment used in this study are listed in the Table of Materials.

1. EV isolation using differential centrifugation

NOTE: A differential centrifugation protocol was employed to isolate EV from human plasma samples. Two ultracentrifugation (UC) cycles with increasing centrifugal force were conducted to maximize purity of the resulting EV pellet.

1. Plasma isolation
   1. Start processing whole blood samples, cooled at 4 °C, within 120 min of collection to ensure plasma EV integrity. Use citrate-supplemented blood collection tubes (0.106 mol/L citrate) to prevent coagulation.
   2. Dilute 9 mL of citrated whole blood 1:1 in ice-cold phosphate-buffered saline (PBS) to reduce viscosity. Add 5 mL of density gradient medium (1.077 g/mL) for separation during centrifugation.
   3. Centrifuge at 600 × g for 25-30 min at 4 °C. Carefully pipette off the plasma fraction from the top of each column into specialized centrifugation tubes. Proceed to the next steps or freeze at -80 °C.
2. Removal of cell debris and apoptotic bodies
   1. Centrifuge the plasma at 20,000 × g for 15 min at 4 °C using an ultracentrifuge with a fixed-angle rotor, without braking. A visible pellet of cell debris and apoptotic bodies should form at the bottom of the tube.
3. EV isolation
   1. Transfer the supernatant to a new tube. Ultracentrifuge at 100,000 × g for 2 h at 4 °C, without braking, to isolate EV, which will form a visible pellet on the tube wall.
   2. Carefully pipette off the supernatant using an electronic pipette, leaving about 1 mL of EV-depleted plasma. Switch to a 1000 µL pipette for the remaining plasma, pipetting slowly to avoid disturbing the EV pellet. Tilt the tube gradually while pipetting to ensure no plasma remains in the tube.
4. Preparation for downstream analysis
   1. Resuspend the isolated EV pellet according to downstream analysis requirements: for nanoparticle tracking, resuspend the EV pellet from 9 mL of whole blood in 200 µL of PBS; for LC-MS/MS, resuspend EV isolated from 36 mL of whole blood in 50 µL of 1x RIPA buffer with 1% protease/phosphatase inhibitor
   2. Transfer the resuspended EV pellet to a 1.5-2 mL tube. Store at -80 °C until further analysis or proceed as needed.

2. Nanoparticle tracking analysis

NOTE: The quantification of average EV size distribution in human plasma samples was conducted using Nanoparticle Tracking Analysis (NTA), a laser-based detection method that analyzes the Brownian motion of nanoparticles.

1. Working dilutions for NTA
   1. Dilute the EV stock solution (EV pellet resuspended in 200 μL PBS) with ice-cold PBS at ratios of 1:10, 1:50, 1:100, and 1:200. Create various working dilutions to determine the most suitable dilution for achieving a final count of 10-100 nanoparticles per frame during nanoparticle tracking.
2. Running NTA
   1. Switch on the NTA instrument and launch the applicable software on the connected computer. Place the chamber inside the laser casket and click on Start Camera to activate it.
   2. Draw 1 mL of PBS into a syringe. Plug the syringe into the front tube and thoroughly flush the tubing system, ensuring no air remains in the chamber. Monitor the camera to detect any air bubbles or contaminating particles.
   3. Draw up the prepared EV working dilution in a 1 mL syringe and inject it into the chamber.
   4. Adjust the Screen Gain to match the monitor's brightness in the Capture and Process tabs. Modify the Camera Level to differentiate nanoparticles clearly on the screen.
   5. Set a suitable Detection Threshold in the Process tab to adjust the sensitivity for differentiating particles from background noise. Keep this value constant for all measurements within the same experiment.
   6. Focus the camera using the wheel on each side of the instrument until the nanoparticles appear sharp on the screen.
   7. Set a constant temperature (ideally 22 °C) for all measurements in the Hardware settings, as Brownian motion is temperature-dependent.
   8. Click on the Run button to start measurement. Enter the sample ID and the solvent in the pop-up window.
   9. Record a minimum of three 30 s videos for each sample run. Advance the sample between measurements to capture a representative sample of the nanoparticle solution. If a syringe pump is available, set it at a constant speed and record continuously.
   10. After recording, the software automatically calculates the concentration and size distribution of nanoparticles. Click on Change to enter the dilution factor used for the sample, and then save the results by clicking on Export.
3. Data analysis
   1. Analyze the results using the output files in a statistical software program.

Label-free analysis of EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

NOTE: Label-free EV proteome analysis was conducted using initial protein separation through gel electrophoresis of EV proteins following membrane lysis. After in-gel protein digestion with trypsin, peptides were analyzed using LC-MS/MS, with individual spectra matched against a human proteome database. Notably, if an untargeted analysis of EV lysates is desired, shotgun proteomics without prior selection of a specific molecular weight range is recommended, making steps 3.1.1-3.1.7 of this protocol unnecessary.

1. In-gel trypsin digestion
   1. Prepare a solution of EV resuspended in 1x RIPA buffer by adding Laemmli loading buffer according to the manufacturer's instructions (see Table of Materials). Boil samples at 95 °C for 5 min.
   2. Load the EV samples, supplemented with loading buffer, along with a protein ladder onto 4%-12% Bis-Tris gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)¹⁴. Stain the gel with Coomassie Blue for 1-4 h to visualize protein bands. De-stain the gel with water by incubating overnight.
   3. Excise Coomassie-stained bands from gel regions containing proteins of interest using a scalpel blade.
   4. Wash the gel pieces with 60 µL of 1:1 (v/v) 50 mM triethylammonium bicarbonate buffer (TEAB) and acetonitrile (ACN), pH 8.5, for 10 min. Shrink the gel pieces three times for 10 min each in 60 µL of ACN. Wash again with 60 µL 50 mM TEAB, pH 8.5.
   5. Reduce the proteins with 10 mM dithiothreitol (DTT) in 100 mM of TEAB at 57 °C for 30 min and dehydrate the gel pieces.
   6. Alkylate the proteins with 10 mM iodoacetamide (IAA) in 100 mM of TEAB at 25 °C for 20 min in the dark.
   7. Wash the gel pieces with 60 µL of 100 mM TEAB and shrink them twice for 10 min each in 60 µL of ACN.
   8. Add 50 mM of TEAB trypsin solution, concentrated accordingly, to the dry gel pieces. Incubate for 4 h at 37 °C. Quench the reaction by adding 20 µL of 0.1% trifluoroacetic acid (TFA).
   9. Extract the resulting peptides by incubating with 30 µL of 1:1 (v/v) 0.1% TFA and ACN for 30 min. Dehydrate the gels with 20 µL of ACN for 20 min, then wash again with 30 µL of 100 mM TEAB for 20 min.
   10. Shrink the gel twice with 20 µL of ACN for 20 min each.
   11. Concentrate the supernatant collected from each extraction step in a vacuum centrifuge and dissolve the sample in 15 µL of 0.1% TFA.
2. LC-MS/MS analysis
   1. Perform nanoflow LC-MS/MS analysis using a high-resolution liquid chromatography mass spectrometer coupled to an electrostatic ion trap analyzer.
   2. Use 0.1% formic acid (FA)/1% ACN as solvent A and 0.1% FA/89.9% ACN as solvent B.
   3. Separate the peptides in a 25-min linear gradient: start from 3% solvent B, increase B to 23% over 21 min, then to 38% over 4 min, and proceed with a washout at 95% B.
   4. Operate the mass spectrometer in data-dependent acquisition mode, automatically alternating between MS and MS2. Acquire MS spectra (m/z 400-1600) with a resolution of 60,000 at m/z 400, generating MS2 spectra for up to 15 precursors using a normalized collision energy of 27 and an isolation width of 1.4 m/z.
3. Protein database search
   1. Search the MS/MS spectra against the Swiss-Prot Homo sapiens (UP000005640, June 2020) protein database and a customized contaminant database (part of MaxQuant, MPI Martinsried^15,16) using Proteome Discoverer 2.5 with Sequest HT.
   2. Configure the program settings as follows: set the fragment ion mass tolerance to 0.02 Da and the parent ion mass tolerance to 5 ppm. Specify trypsin as the enzyme used for digestion, and set carbamidomethyl as a fixed modification for cysteine, with oxidation (methionine) and deamidation (asparagine, glutamine) as variable modifications. Include acetylation, methionine loss, and their combination as variable modifications of the protein terminus.
   3. Quantify peptides using the precursor ion quantifier mode, employing the Top N Average (n = 3) method for protein abundance calculation¹⁷.

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

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