May 11, 2026

Isolation, characterization, and proteomic analysis of plasma-derived exosomes and microvesicles in Parkinson’s disease

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3mj17l25/v1
  • Wenzheng Hu1,2,3,
  • Lingxiao Cao2,3,4,
  • Duo Wang5,
  • Weiwei Cao2,
  • Qing Yang2,
  • Zhirong Wan6,
  • Yue Huang2,3,7,
  • Pingyi Xu1
  • 1Department of Neurology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China;
  • 2Human Brain and Tissue Bank, China National Clinical Research Center for Neurological Diseases, Beijing Tiantan Hospital, Capital Medical University, Beijing, China;
  • 3Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China;
  • 4Department of Neurology, The Second Qilu Hospital of Shandong University, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China;
  • 5Department of Neurology, Hebei University Affiliated Hospital, Clinical Medical College, Baoding, Hebei, China;
  • 6Department of Neurology, Aerospace Central Hospital, Beijing, China;
  • 7Centre for Healthy Brain Aging (CHeBA), Discipline of Psychiatry and Mental Health, School of Clinical Medicine, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia
Wenzheng Hu
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3mj17l25/v1
Protocol Citation: Wenzheng Hu, Lingxiao Cao, Duo Wang, Weiwei Cao, Qing Yang, Zhirong Wan, Yue Huang, Pingyi Xu 2026. Isolation, characterization, and proteomic analysis of plasma-derived exosomes and microvesicles in Parkinson’s disease. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mj17l25/v1
License: This is an open access  protocol  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
Protocol status: Working
We use this protocol and it's working
Created: May 07, 2026
Last Modified: May 11, 2026
Protocol  Integer ID: 316620
Keywords: proteomic, exosomes, microvesicles, extracellular vesicles, plasma, parkinson’s disease, ultracentrifugation, isolation, characterization, potential biomarkers for pd, derived exosome, microvesicles in parkinson, disease extracellular vesicle, extracellular vesicle, including exosome, proteomic analysis on the isolated mv, potential biomarker, proteomic profiles of plasma, proteomic analysis of plasma, proteomic analysis on mv, proteomic profile, proteomic analysis, performing proteomic analysis, biomarker, protein marker, valuable sources of biomarker, exo from the plasma, particle sizes of exo, plasma of pd patient, parkinson, mvs in pd patient, using nanoparticle
Abstract
Extracellular vesicles (EVs), including exosomes (Exo) and microvesicles (MVs), have emerged as valuable sources of biomarkers. While Exo have been extensively studied in Parkinson's disease (PD), research on MVs remains limited. Investigating the proteomic profiles of plasma-derived MVs in PD patients compared to control subjects may help identify potential biomarkers for PD. This protocol details a workflow for isolating MVs and Exo from the plasma of PD patients and control subjects, characterizing these EVs using nanoparticle tracking analysis (NTA), Western blot, and electron microscopy, and subsequently performing proteomic analysis on the isolated MVs. Specifically, Exo and MVs are extracted from plasma via ultracentrifugation. The particle sizes of Exo and MVs are determined by NTA, while their protein markers are evaluated by Western blot: CD9, CD63, and CD81 serve as positive EVs markers, APOA1 as a negative EVs marker, and Actinin‑4 as an MVs‑enriched marker. The morphology and size of these EVs are further examined by negative‑staining electron microscopy. Finally, Direct Data‑Independent Acquisition (DIA) technology is employed to conduct proteomic analysis on MVs isolated from the plasma of PD patients and normal control subjects.
Guidelines
  1. Avoid repeated freeze-thaw cycles; multiple cycles will degrade EVs in the plasma.
  2. All ultracentrifugation steps must be performed at 4°C.
  3. When samples are not being centrifuged, keep them on ice at all times.
  4. To resuspend MVs or Exo pellets, thoroughly pipette the pellet area at the bottom of the centrifuge tube with at least 50 pipetting strokes.
  5. If EVs are to be used for NTA or electron microscopy, resuspend them in PBS; for Western blot or proteomic analysis, resuspend in RIPA lysis buffer.
Safety warnings
  1. Samples and reagents taken out of the -80°C freezer may cause frostbite. Anti-freezing gloves must be worn during handling to avoid cold-related injuries.
  2. Human plasma should be treated as potentially infectious (e.g., HIV, HBV, etc.). Appropriate protective measures should be taken, including wearing laboratory gloves, a lab coat, and safety goggles.
  3. Before centrifugation using an ultracentrifuge, all centrifuge tubes must be weighed and balanced to ensure the weight difference is less than 0.005 grams, so as to prevent damage to the rotor.

Ethics statement
This study was approved by the Institutional Ethics Committee and was conducted in accordance with the Declaration of Helsinki. All enrolled subjects provided written informed consent.
Before start
  1. If the plasma used for EV isolation has been stored at -80°C, thaw it at 4°C before starting the experiment.
  2. Prior to the ultracentrifugation steps for MVs and Exo separation (20,000×g or 150,000×g), pre-cool the centrifuge rotor to 4°C.
  3. Use sterile PBS that has been filtered through a 0.22 µm filter and pre-cooled to 4°C.
  4. Before NTA measurement, ensure that the NTA instrument has been cleaned with particle‑free water.
Plasma Collection
30m
Five milliliters of venous blood were collected from each fasting participant in the morning.
Separate plasma by centrifugation at 2,500×g for 15 minutes, carefully avoiding the buffy coat layer.
15m
Transfer the plasma supernatant to a new 10 mL centrifuge tube and centrifuge again at 2,500×g for 15 minutes to remove residual cell debris and remaining platelets.
15m
Aliquot the resulting plasma into cryovials (1 mL per tube) and store at –80°C until use.
Isolation of microvesicles from plasma
3h 20m
One milliliter of plasma was transferred into a 1.5 mL centrifuge tube and centrifuged at 2,500×g for 15 minutes to remove dead cells and cell debris.
15m
The supernatant was transferred into a new tube and centrifuged at 17,000×g for 90 minutes at 4°C using a refrigerated centrifuge (Thermo Scientific Fresco 21) to obtain a microvesicles (MVs) pellet.
1h 30m
After centrifugation, the supernatant was discarded, unless exosomes (Exo) are required, in which case the supernatant from this step should be collected for subsequent Exo isolation (see the isolation of exosomes from plasma section). The MVs pellet was then resuspended in 500 μL PBS and centrifuged again at 17,000×g for 90 minutes at 4 °C using a refrigerated centrifuge (Thermo Scientific Fresco 21).
1h 30m
The supernatant was aspirated, and the tube was then inverted on a paper towel for 3–5 minutes to remove residual supernatant.
5m
The final MVs pellet was resuspended in 50–100 μL of PBS or RIPA lysis buffer, depending on downstream applications.
Isolation of exosomes from plasma
1h 35m
The supernatant from step 6 was filtered through a 0.22 μm filter into a 10.4 mL ultracentrifuge tube (Thermo Fisher, catalog #010-1382), balanced with PBS, and then ultracentrifuged using a Beckman Optima XPN-90 ultracentrifuge with a Thermo ScientificTM FiberliteTM F65L‑6 × 13.5 rotor at 150,000×g for 90 minutes at 4°C to obtain an Exo pellet.
1h 30m
After centrifugation, the supernatant was discarded. The tube was then inverted on a paper towel for 3–5 minutes to remove residual supernatant.
5m
The Exo pellet was resuspended in 50 μL of PBS or RIPA buffer, depending on downstream applications.
Western Blot Analysis of Extracellular Vesicles Markers
19h 47m 30s
Protein Quantification and Sample Preparation

The protein concentration of each Extracellular vesicles (EVs) preparation was determined using the micro bicinchoninic acid (BCA) protein assay according to the manufacturer’s instructions (Cat. No. B0010-1, Beyotime).
Take 20 µg of protein per sample.
Add an appropriate amount of 5× concentrated buffer to each sample to adjust the final concentration to 1× . Then, add 1× buffer to each sample to equalize the volume of all samples.
Heat the samples at 95°C for 10 minutes to denature proteins.
10m
Briefly centrifuge the samples to collect condensation.
SDS-PAGE (Electrophoresis)
Load the denatured samples onto an 8–16% gradient SDS-polyacrylamide gel.
Run the gel at constant voltage: 80 V for 10 minutes (stacking gel), then increase to 120 V for 1 hour (separating gel).
1h 10m
Protein Transfer to PVDF Membrane
Activate PVDF membrane in methanol for 15–30 seconds, then rinse in transfer buffer.
30s
Assemble the transfer sandwich (gel against PVDF membrane).
Transfer proteins to the PVDF membrane using a wet transfer apparatus at constant current 200 mA for 2 hours.
2h
Blocking
After transfer, wash the membrane briefly in 1× TBST.
Block the membrane with 5% skimmed milk in 1× TBST at room temperature for 45 minutes with gentle shaking.
45m
Wash the membrane with 1× TBST for 10 minutes at room temperature with shaking, and repeat this washing step two more times for a total of three 10-minute washes.
30m
Primary Antibody Incubation
Dilute primary antibodies in blocking buffer or TBST as follows: CD9 (Abcam, ab223052) – 1:2000, CD63 (ImmunoWay, YT5525) – 1:2000, CD81 (Biolegend, 349501) – 1:2000, Actinin-4 (Abcam, ab108198) – 1:2000, APOA1 (Abcam, ab52945) – 1:2000. Incubate the membrane with primary antibody solution overnight at 4°C with gentle shaking.
12h
Remove the primary antibody solution.
Wash the membrane with 1× TBST for 10 minutes at room temperature with shaking, and repeat this washing step two more times for a total of three 10-minute washes.
30m
Secondary Antibody Incubation
Dilute HRP-conjugated secondary antibody (anti-rabbit or anti-mouse, depending on the primary host species) at 1:5000 in 1× TBST, and incubate the membrane with the solution for 2 hours at room temperature with gentle shaking.
2h
Remove the secondary antibody solution.
Wash the membrane three times with 1× TBST at room temperature with shaking: the first two washes for 10 minutes each, and the third wash for 20 minutes.
40m
Signal Detection
Prepare enhanced chemiluminescence (ECL) substrate according to the manufacturer’s instructions.
Drain excess liquid from the membrane (do not let it dry).
Apply ECL substrate to the membrane and incubate for 1–2 minutes.
2m
Visualize chemiluminescent signals using a gel documentation system (e.g., ChemiDoc).
Electron Microscopy Protocol for Morphological Analysis of Extracellular Vesicles
5m
Take a small amount of EVs sample and dilute it 100-fold with PBS (e.g., 3 µL EV + 297 µL PBS).
Drop an appropriate volume (e.g., 5–10 µL) of the diluted EVs sample onto a copper grid coated with Lacey carbon film.
Add 20 µL of 2% phosphotungstic acid to the grid and incubate for 5 minutes.
5m
Carefully remove excess staining solution and allow the grid to air-dry completely.
Examine the dried grid under a transmission electron microscope (e.g., Tecnai‑G2‑Spirit‑FEI/Quanta Microscope; Philips, Eindhoven, Netherlands). Capture images for morphological analysis.
NTA Protocol for Concentration and Size Analysis of Extracellular Vesicles
Prepare instrument
Calibrate the ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) using 100 nm polystyrene beads (ThermoFisher Scientific) prior to sample analysis.
Flush the sample chamber with sterile PBS that has been filtered through a 0.22 μm filter to remove any residual particles. Ensure the chamber is completely clean before sample loading.
Sample dilution and loading
Dilute each EVs sample 1000‑fold in sterile 0.22 μm filtered PBS (e.g., 2 µL sample into 1998ul PBS). Mix gently.
Inject the diluted sample into the sample chamber.
Parameter setting and measurement
Set pre‑acquisition parameters: temperature 23°C, sensitivity 70, specificity 70, frame rate 30 fps, shutter speed 100.
Measure at 11 different positions, with 3 cycles per position.
Apply post‑acquisition parameters: minimum brightness 25, particle size range 5–1000 nm.
Analyze data
Use ZetaView software (version 8.02.28) and Microsoft Excel 2013 for data analysis.
Proteomics Analysis
17h 15m
Extraction of Sample Proteins
MVs from the PD group and the control group were extracted following the method described in the Isolation of microvesicles from plasma section.
Protein Concentration Measurement
Protein concentration for each sample was measured using the BCA method as described in the Protein Quantification and Sample Preparation of the Western Blot Analysis of Extracellular Vesicle Markers section.
Electrophoresis of Equal Protein Mass Samples
The steps were performed in the same manner as described in the Western blot electrophoresis procedure.
Filter-Aided Sample Preparation (FASP) for Protein Digestion
Protein Reduction：
Equal amounts of protein from each sample were added to 10 kDa ultrafiltration tubes. The sample liquids were replaced with lysis buffer, followed by centrifugation to achieve equal volumes. Dithiothreitol (DTT) was added at a ratio of 40:1 (final DTT concentration 25 mM) relative to the total solution volume. The mixture was vortexed, then incubated in a water bath at 37°C for 1 hour. The samples were cooled to room temperature.
1h
Protein Alkylation: Iodoacetamide (IAA) was added to the reduced samples at a solution-to-IAA ratio of 20:1 (final IAA concentration 50 mM). The mixture was vortexed, then incubated at room temperature in the dark for 30 minutes.
30m
Sample Washing: A 5-fold dilution of TEAB (300 µl) was added to the samples, followed by centrifugation at 12,000 rpm (13,400 g) for 10 minutes. The solution at the bottom of the collection tube was discarded. This process was repeated three times. Subsequently, a 2-fold dilution of TEAB (300 µl) was added, and the centrifugation step was repeated.
1h
Enzymatic Digestion in Filter-Aided Solution: The ultrafiltration tube was replaced with a new collection tube. Trypsin was added at a protein-to-trypsin ratio of 15:1 and incubated at 37°C overnight for digestion.
12h
Peptide Collection:
On the following day, a 2-fold dilution of TEAB (100 µl) was added, followed by centrifugation at 12,000 rpm (13,400 g) for 10 minutes. This process was repeated three times. The peptide solution obtained from enzymatic digestion was collected at the bottom of the tube.
40m
Freeze-Dried Samples Place the collected eluent in a rotary vacuum concentrator and vacuum dry it without heating. After drying, store it in a -80°C freezer for future use.
C18 Solid-Phase Extraction
Activation of the C18 Solid-Phase Extraction Column: Pipette 100 µL of 100% ACN into the column, centrifuge at 3,000 rpm for 2 minutes.
2m
Equilibration of the C18 Solid-Phase Extraction Column: Pipette 100 µL of 50% ACN with 0.1% FA into the column, centrifuge at 3,000 rpm for 3 minutes.
3m
 Second Equilibration of the C18 Solid-Phase Extraction Column:
Pipette 100 µL of 2% ACN with 0.1% FA into the column, centrifuge at 3,000 rpm for 3 minutes.
3m
Sample Loading:
Redissolve the sample in 50 µL of 0.1% TFA and load it into the column. Centrifuge at 2,700 rpm for 3 minutes, repeating this process three times.
12m
Desalting Wash: Pipette 100 µL of 2% ACN with 0.1% FA, centrifuge at 3,000 rpm for 3 minutes, and repeat this process twice.
9m
Elution: Pipette 50 µL of 50% ACN with 0.1% FA, collect the eluate into an EP tube, and centrifuge at 2,700 rpm for 3 minutes. Repeat this process once and combine the eluates.
6m
Freeze-Dry the Eluate: Place the collected eluate in a rotary vacuum concentrator and vacuum dry it without heating. After drying, store it in a -80°C freezer for future use.
LC-MS/MS Analysis
 Redissolve the peptide fractions in 10 µL of 0.1% FA solution.
Perform separation using a Vanquish liquid chromatography system with a nanoViper C18 column (75 μm × 250 mm, 2 μm). The mobile phases are as follows:Phase A: 0.1% FA in water, Phase B: 80% acetonitrile with 0.1% FA in water. The effective elution gradient is 1%–35% over a total elution time of 90 minutes, with a flow rate of 0.6 µL/min.The liquid chromatography conditions are as follows:
Low-pH Reversed-Phase Chromatography Gradient
Time (min)A%B%Flow Rate (nL/min)
00:009646600
78:006535600
81:004060600
84:009010600
90:009010600

Perform mass spectrometry analysis
Use an Orbitrap Q-Exactive HF mass spectrometer in high-sensitivity mode. Set full scan (MS1): resolution 120,000, scan range 350–1250 m/z, AGC target 3e6, max injection time 50 ms. Set fragmentation scan (MS2): resolution 30,000, AGC target 1e6, collision energy 25%/30%/35%. Configure DIA with 80 scanning windows. Total scan time: 90 minutes.
1h 30m
DIA Library Search
Process spectral data
Use Spectronaut (version 16.0.220606.53000) in directDIA mode with the reference database uniprot_human_81803_20230327.fasta.

.
Set search parameters
Use human protein database; enzyme: trypsin (up to 2 missed cleavages); precursor mass tolerance ±10 ppm; fragment mass tolerance ±0.02 Da; fixed modification: carbamidomethylation (C); variable modifications: oxidation (M) and N-terminal acetylation; FDR <1% at peptide and protein level; protein identification requires ≥1 unique peptide.
DIA Data Import
Import DIA data into Spectronaut and retain results with a q-value <1.0% for final analysis.
Protocol references
[1]Pedersen S, Jensen KP, Honoré B, et al. Circulating microvesicles and exosomes in small cell lung cancer by quantitative proteomics. Clin Proteomics. 2022;19(1):2. Published 2022 Jan 7. doi:10.1186/s12014-021-09339-5
[2]Dorado E, Doria ML, Nagelkerke A, et al. Extracellular vesicles as a promising source of lipid biomarkers for breast cancer detection in blood plasma. J Extracell Vesicles. 2024;13(3):e12419. doi:10.1002/jev2.12419
[3]Kowal J, Arras G, Colombo M, et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc Natl Acad Sci U S A. 2016;113(8):E968-E977. doi:10.1073/pnas.1521230113
[4]Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750. Published 2018 Nov 23. doi:10.1080/20013078.2018.1535750