The authors thank Heidi Deigentasch, Amelie Werner, and Elisabeth Mertz for their organizational support during this project.

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 &#176;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.
<ol>
	<li>Plasma isolation
	<ol>
		<li>Start processing whole blood samples, cooled at 4 &#176;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.</li>
		<li>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.</li>
		<li>Centrifuge at 600 &#215; g for 25-30 min at 4 &#176;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 &#176;C.</li>
	</ol>
	</li>
	<li>Removal of cell debris and apoptotic bodies
	<ol>
		<li>Centrifuge the plasma at 20,000 &#215; g for 15 min at 4 &#176;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.</li>
	</ol>
	</li>
	<li>EV isolation
	<ol>
		<li>Transfer the supernatant to a new tube. Ultracentrifuge at 100,000 &#215; g for 2 h at 4 &#176;C, without braking, to isolate EV, which will form a visible pellet on the tube wall.</li>
		<li>Carefully pipette off the supernatant using an electronic pipette, leaving about 1 mL of EV-depleted plasma. Switch to a 1000 &#181;L 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.</li>
	</ol>
	</li>
	<li>Preparation for downstream analysis
	<ol>
		<li>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 &#181;L of PBS; for LC-MS/MS, resuspend EV isolated from 36 mL of whole blood in 50 &#181;L of 1x RIPA buffer with 1% protease/phosphatase inhibitor</li>
		<li>Transfer the resuspended EV pellet to a 1.5-2 mL tube. Store at -80 &#176;C until further analysis or proceed as needed.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Working dilutions for NTA
	<ol>
		<li>Dilute the EV stock solution (EV pellet resuspended in 200 &#956;L 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.</li>
	</ol>
	</li>
	<li>Running NTA
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Draw up the prepared EV working dilution in a 1 mL syringe and inject it into the chamber.</li>
		<li>Adjust the Screen Gain to match the monitor&#39;s brightness in the Capture and Process tabs. Modify the Camera Level to differentiate nanoparticles clearly on the screen.</li>
		<li>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.</li>
		<li>Focus the camera using the wheel on each side of the instrument until the nanoparticles appear sharp on the screen.</li>
		<li>Set a constant temperature (ideally 22 &#176;C) for all measurements in the Hardware settings, as Brownian motion is temperature-dependent.</li>
		<li>Click on the Run button to start measurement. Enter the sample ID and the solvent in the pop-up window.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Analyze the results using the output files in a statistical software program.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>In-gel trypsin digestion
	<ol>
		<li>Prepare a solution of EV resuspended in 1x RIPA buffer by adding Laemmli loading buffer according to the manufacturer&#39;s instructions (see Table of Materials). Boil samples at 95 &#176;C for 5 min.</li>
		<li>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.</li>
		<li>Excise Coomassie-stained bands from gel regions containing proteins of interest using a scalpel blade.</li>
		<li>Wash the gel pieces with 60 &#181;L 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 &#181;L of ACN. Wash again with 60 &#181;L 50 mM TEAB, pH 8.5.</li>
		<li>Reduce the proteins with 10 mM dithiothreitol (DTT) in 100 mM of TEAB at 57 &#176;C for 30 min and dehydrate the gel pieces.</li>
		<li>Alkylate the proteins with 10 mM iodoacetamide (IAA) in 100 mM of TEAB at 25 &#176;C for 20 min in the dark.</li>
		<li>Wash the gel pieces with 60 &#181;L of 100 mM TEAB and shrink them twice for 10 min each in 60 &#181;L of ACN.</li>
		<li>Add 50 mM of TEAB trypsin solution, concentrated accordingly, to the dry gel pieces. Incubate for 4 h at 37 &#176;C. Quench the reaction by adding 20 &#181;L of 0.1% trifluoroacetic acid (TFA).</li>
		<li>Extract the resulting peptides by incubating with 30 &#181;L of 1:1 (v/v) 0.1% TFA and ACN for 30 min. Dehydrate the gels with 20 &#181;L of ACN for 20 min, then wash again with 30 &#181;L of 100 mM TEAB for 20 min.</li>
		<li>Shrink the gel twice with 20 &#181;L of ACN for 20 min each.</li>
		<li>Concentrate the supernatant collected from each extraction step in a vacuum centrifuge and dissolve the sample in 15 &#181;L of 0.1% TFA.</li>
	</ol>
	</li>
	<li>LC-MS/MS analysis
	<ol>
		<li>Perform nanoflow LC-MS/MS analysis using a high-resolution liquid chromatography mass spectrometer coupled to an electrostatic ion trap analyzer.</li>
		<li>Use 0.1% formic acid (FA)/1% ACN as solvent A and 0.1% FA/89.9% ACN as solvent B.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Protein database search
	<ol>
		<li>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 Martinsried15,16) using Proteome Discoverer 2.5 with Sequest HT.</li>
		<li>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.</li>
		<li>Quantify peptides using the precursor ion quantifier mode, employing the Top N Average (n = 3) method for protein abundance calculation17.</li>
	</ol>
	</li>
</ol>

Cardiovascular Biomarker Studies Using Plasma Extracellular Vesicle Proteome

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V., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-gel+digestion+for+mass+spectrometric+characterization+of+proteins+and+proteomes.">In-gel digestion for mass spectrometric characterization of proteins and proteomes.</a> Nat Protoc. 1 (6), 2856-2860 (2006).</li><li>Bateman, N. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+peptide+identification+events+in+proteomic+workflows+using+data-dependent+acquisition+(dda).">Maximizing peptide identification events in proteomic workflows using data-dependent acquisition (dda).</a> Mol Cell Proteomics. 13 (1), 329-338 (2014).</li><li>Pino, L. K., Just, S. C., Maccoss, M. J., Searle, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquiring+and+analyzing+data+independent+acquisition+proteomics+experiments+without+spectrum+libraries.">Acquiring and analyzing data independent acquisition proteomics experiments without spectrum libraries.</a> Mol Cell Proteomics. 19 (7), 1088-1103 (2020).</li></ol>

EG received honoraria for lecturers from Roche Diagnostics, BRAHMS Thermo Scientific, Bayer Vital GmbH, AstraZeneca, Lilly Deutschland, Boehringer Ingelheim; he received institutional research grants from Roche Diagnostics and Daiichi Sankyo, and serves as a consultant for Roche Diagnostics, BRAHMS Thermo Scientific, Astra Zeneca, Novartis and Boehringer Ingelheim, outside the submitted work. JBK received project-related funding from the German Centre for Cardiovascular Research (DZHK) and Roche Diagnostics. The remaining authors declare no conflict of interest related to the submitted work.

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...

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 EV5,6,7. 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 diseases8,9,10. 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 relevance6,11.
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.

<table><tbody><tr><td>4x Laemmli sample bufffer</td><td>Bio-rad</td><td>1610747</td><td></td></tr><tr><td>10x RIPA-Buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>26.3 mL Polycarbonate Bottle with Cap Assembly for Ultracentrifugation</td><td>Beckman Coulter</td><td>355654</td><td></td></tr><tr><td>5810R Benchtop centrifuge</td><td>Eppendorf</td><td>5811000015</td><td></td></tr><tr><td>Acetonitrile (ACN)</td><td>Biosolve</td><td>0001204101BS</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>43816-10ML</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Formic Acid 99% ULC/MS 100</td><td>Biosolve</td><td>0006914143BS</td><td></td></tr><tr><td>Histopaque - 1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Iodoacetamide (IAA)</td><td>Sigma-Aldrich</td><td>I6125-5G</td><td></td></tr><tr><td>Mass Spectrometer Orbitrap Q Exactive HF</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>MOPS SDS running buffer</td><td>Thermo Fisher</td><td>B0001</td><td></td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical&amp;nbsp;</td><td>NS300</td><td></td></tr><tr><td>Nanosight NTA 3.2 Software</td><td>Malvern Panalytical&amp;nbsp;</td><td></td><td></td></tr><tr><td>NuPAGE 4&amp;nbsp;bis 12&amp;nbsp;%, Bis-Tris protein gels</td><td>Invitrogen</td><td>NP0323</td><td></td></tr><tr><td>Optima XPN-80 floor-standing ultracentrifuge</td><td>Beckman Coulter</td><td>521-4180</td><td></td></tr><tr><td>PageRuler Plus Prestained Protein Ladder</td><td>Thermo Fisher Scientific</td><td>26619</td><td></td></tr><tr><td>Protease/Phosphatase Inhibitor Cocktail (100X)</td><td>Cell Signaling Technology</td><td>5872S</td><td></td></tr><tr><td>Protein marker</td><td>Thermo Fisher</td><td>26616</td><td></td></tr><tr><td>Proteome Discoverer 2.5</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Quick stain coomasssie</td><td>Serva</td><td>35081.01</td><td></td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 1.9 &amp;micro;m 1 g</td><td>Dr. Maisch</td><td>r119.aq.0001</td><td></td></tr><tr><td>SDS-PAGE commercial gel</td><td>Thermo Fisher</td><td>NW00100BOX</td><td></td></tr><tr><td>S-Monovette&amp;nbsp;Citrat 9NC 0.106 mol/l 3,2%</td><td>Sarstedt</td><td>02.1067.001</td><td></td></tr><tr><td>Speed vac concentrator</td><td>Savant</td><td></td><td></td></tr><tr><td>Swiss-Prot (Uniprot) Homo sapiens (UP000005640, June 2020) protein database</td><td>UniProt</td><td>https://www.uniprot.org/proteomes/UP000005640</td><td></td></tr><tr><td>Triethylammonium bicarbonate buffer (TEAB)</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA) for HPLC &gt;99%, 100 mL</td><td>Sigma-Aldrich</td><td>302031-100ML</td><td></td></tr><tr><td>Trypsin MS-Grade</td><td>Thermo Fisher</td><td>90058</td><td></td></tr><tr><td>Type 50.2 Ti Fixed-Angle Rotor</td><td>Beckman Coulter</td><td>337901</td><td></td></tr><tr><td>UHPLC Dionex Ultimate 3000</td><td>Thermo Fisher Scientific</td><td>ULTIM3000RSLCNANO</td><td></td></tr><tr><td>Water</td><td>Biosolve</td><td>0023214102BS</td><td></td></tr></tbody></table>

isolation, characterization, and proteomic analysis of plasma-derived extracellular vesicles for cardiovascular biomarker discovery

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.

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, ...

Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies

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).

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

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

isolation-characterization-proteomic-analysis-plasma-derived

Research

JoVE Journal

Medicine

316 Views.  University Hospital Heidelberg. Circulerende extracellulaire blaasjes als biomarkers zijn een relatief nieuw veld in het onderzoek naar biomarkers met een enorm potentieel. Ons protocol is bedoeld om een praktische benadering te bieden voor het implementeren van extracellulaire vesikel gebonden biomarker ontdekking en detectie in een spoedeisende hulpafdeling. Opkomend bewijs heeft aangetoond dat de moleculaire lading die in extracellulaire blaasjes en bloedsomloop wordt aangetroffen, eiwit...

Video: Auteur Spotlight: Bevordering van de analyse van plasma extracellulair blaasjesproteoom voor cardiovasculaire biomarkerstudies

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Biochemistry

Enhancing EV Isolation for Precise Biofluid Analysis

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

Author Spotlight: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Immunology and Infection

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Neuroscience

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Biology

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Efficient Recycling of Polycarbonate Ultracentrifuge Tubes for Proteomics

Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins.
This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples.
Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (&#8804;20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Author Spotlight: Effective Reuse of Polycarbonate Tubes for Extracellular Vesicle Isolation

A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ...

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

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