Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Jiale Cheng; Jiong Zhu; Yujiao Liu; Chen Yang; Yuehui Zhang; Yuchen Liu; Chaozhi Jin; Jian Wang

doi:10.3791/65521

JoVE Journal > Biochemistry

生化学

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages

Published: June 30, 2023

doi:

10.3791/65521

Jiale Cheng^1,2, Jiong Zhu^1,2, Yujiao Liu^2,3, Chen Yang^2,3, Yuehui Zhang², Yuchen Liu², Chaozhi Jin², Jian Wang^1,2

¹School of Basic Medical Sciences,Anhui Medical University, ²State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing),Beijing Institute of Lifeomics, ³School of Life Sciences,Hebei University

概要

This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Abstract

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of <200 nm are present in various body fluids. These sEVs regulate various biological processes such as gene transcription and translation, cell proliferation and survival, immunity and inflammation through their cargos, such as proteins, DNA, RNA, and metabolites. Currently, various techniques have been developed for sEVs isolation. Among them, the ultracentrifugation-based method is considered the gold standard and is widely used for sEVs isolation. The peptides are naturally biomacromolecules with less than 50 amino acids in length. These peptides participate in a variety of biological processes with biological activity, such as hormones, neurotransmitters, and cell growth factors. The peptidome is intended to systematically analyze endogenous peptides in specific biological samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Here, we introduced a protocol to isolate sEVs by differential ultracentrifugation and extracted peptidome for identification by LC-MS/MS. This method identified hundreds of sEVs-derived peptides from bone marrow-derived macrophages.

Introduction

Small extracellular vesicles (sEVs) with a diameter of less than 200 nm are present in almost all types of body fluids and secreted by all kinds of cells, including urine, sweat, tears, cerebrospinal fluid, and amniotic fluid¹. Initially, sEVs were considered as receptacles for disposing of cellular waste, which led to minimal research in the subsequent decade². Recently, increasing evidence indicates that sEVs contain specific proteins, lipids, nucleic acids, and other metabolites. These molecules are transported to target cells³, contributing to intercellular communication, through which they participate in various biological processes, such as tissue repair, angiogenesis, immunity⁴ and inflammation⁵^,⁶, tumor development and metastasis⁷^,⁸^,⁹, etc.

To facilitate the study of sEVs, it is imperative to isolate sEVs from complex samples. Different sEVs isolation methods have been developed based on the physical and chemical properties of sEVs, such as their density, particle size, and surface marker proteins. These techniques include ultracentrifugation-based methods, particle size-based methods, immunoaffinity capture-based methods, sEVs precipitation-based methods, and microfluidics-based methods¹⁰^,¹¹^,¹². Among these techniques, the ultracentrifugation-based method is widely recognized as the gold standard for sEVs isolation and is the most commonly used technique¹³.

An increasing amount of evidence suggests the presence of a multitude of undiscovered biologically active peptides in the peptidomes of various organisms. These peptides significantly contribute to numerous physiological processes by regulating growth, development, stress response¹⁴^,¹⁵, and signal transduction¹⁶. The objective of sEVs' peptidome is to uncover the peptides carried by these sEVs and provide clues to their biological functions. Here, we present a protocol of isolating sEVs through differential ultracentrifugation, followed by extraction of peptides from these sEVs for further analysis of their peptidome.

Protocol

1. Isolation of small extracellular vesicles

NOTE: Perform all centrifugation in steps 1.1-1.11 at 4 °C.

Preparation of sEVs-free fetal bovine serum (FBS): Centrifuge FBS overnight at 110,000 × g at 4 °C through an ultracentrifuge (see Table of Materials) to remove endogenous sEVs. Collect the supernatant, filter sterilize it with a 0.2 µm ultrafiltration membrane, and store it at -20 °C.
Plate about 3 x 10⁷ immortalized bone marrow-derived macrophages (iBMDMs) on a 150 mm culture dish and add 20 mL of DMEM culture medium (see Table of Materials).
Before collecting sEVs, discard the medium. Wash the cells once with PBS (phosphate-buffered saline; Table of Materials) and replace it with a medium containing 10% sEVs-free FBS.
According to the needs of the experiment, collect the cell supernatant and transfer it to 50 mL centrifuge tubes.
Centrifuge the cell supernatant at 300 × g for 10 min to remove the cells, discard the pellet, and transfer the supernatant to new 50 mL centrifuge tubes.
Centrifuge the supernatant at 2000 × g for 10 min to remove dead cells, discard the pellet, and transfer the supernatant to new high-speed centrifuge tubes (see Table of Materials).
Centrifuge the supernatant at 10,000 × g for 30 min through a high-speed centrifuge to remove cell debris and microvesicles, discard the pellet, and transfer the supernatant to new ultracentrifuge tubes (see Table of Materials). The volume of the open ultracentrifuge tubes is 38.6 mL; place 35 mL of cell supernatant in each tube.
Centrifuge the supernatant at 110,000 × g for 70 min in a swinging-bucket rotor centrifuge(see Table of Materials) to obtain crude sEV pellet.
Discard the supernatant, and wash the sEVs-enriched pellet with 1 mL of PBS. Centrifuge at 110,000 × g for 70 min on a Tabletop ultracentrifuge (see Table of Materials). Discard the supernatant and add 1 mL of PBS to an ultracentrifuge tube.
Then pipette the precipitate continuously, transfer the 1 mL of PBS to another ultracentrifuge tube, and so on, until all ultracentrifuge tubes are mixed by pipetting.
Discard the supernatant, and resuspend the pellet in 100 µL of PBS, which is sEVs.
Measure the total protein of sEVs using the Bicinchoninic Acid (BCA) method, following the steps below (see Table of Materials).
1. Preparation of protein standard: Add 1.2 mL of protein preparation solution into a standard protein tube (30 mg of BSA) to fully dissolve it to prepare protein standard solution (25 mg/mL). Then dilute it with PBS to a final concentration of 0.5 mg/mL.
2. Add 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL of the protein standard into the 96-well plate, and add PBS to make up 20 µL. At the same time, add 18 µL of HEPES lysis buffer (20 mM HEPES, 50 mM NaCl, 1 mM NaF, 0.5% Triton X-100) and 2 µL of sEVs samples to the sample wells to be tested.
3. Prepare the required BCA working solution as per the manufacturer's instructions, add 200 µL of BCA working solution to each well, and place at room temperature (RT) for 20-30 min.
4. Measure the absorbance at 562 nm wavelength with a microplate reader, and calculate the total protein concentration of sEVs according to the standard curve and the dilution factor.
  NOTE: The sEVs extracted from 40 mL of supernatant by this protocol can be used for subsequent identification of protein markers (western blot). However, sEVs extracted from 200 mL of cell supernatant can be used for both morphological observation (transmission electron microscopy, TEM) and particle size analysis (nanoparticle tracking analysis, NTA). Specifically, for sEVs harvested in step 1.11, resuspend with 100 µL of PBS. Take 20-30 µL for morphology observation (TEM) and resuspend the remaining sample in 1 mL PBS for particle size analysis (NTA). All centrifuges are pre-cooled in advance. For step 1.8, these ultracentrifuge tubes must be strictly balanced and at least three-quarters full. If not, add medium to makeup. For step 1.11, the sEVs can be stored at 4 °C for a short term (12 h); otherwise, they must be stored at -20 or -80 °C to avoid protein degradation that interferes with the extraction of peptides. However, for samples used to observe the morphology and measure particle size, it is only recommended to store at 4 °C for a short term (12 h).

2. Observation of morphology of sEVs by transmission electron microscopy

Place a drop of fresh sEVs sample (about 20-30 µL) on the parafilm, place the number side of the copper mesh on the sEVs droplet, and let it absorb for 3 min.
Absorb the excess liquid with filter paper, and then place the copper grid on a drop of distilled water and wash it twice.
Absorb the excess liquid with filter paper. Then place the copper grid on the 0.5% uranyl acetate droplet for negative staining for 5 s.
Repeat step 2.2.
Place the prepared copper mesh on the sample rod and insert it into the sample stage.
1. Adjust the magnification to 20,000x-25,000x to find the sample to be tested. Then adjust the magnification to 100,000x and adjust the appropriate position and grayscale to observe under a transmission electron microscope (TEM; Table of Materials). The accelerating voltage for acquiring images is set at 80 kV.
  NOTE: For step 2.1, if the concentration of sEVs samples is low (<50 µg/µL), the adsorption time can be extended to 5 min or even longer. Alternatively, for step 1.10, a smaller volume of PBS (50 µL) can be used for resuspension. For step 2.3, the negative staining time should not be too long; otherwise, it is difficult to observe the three-dimensional (3D) structure of sEVs.

3. Measurements of particle size distribution and concentration of sEVs by nanoparticle tracking analysis

For the sEVs harvested in step 1.11, dilute the solution to 1 mL for nanoparticle tracking analysis (NTA). First, clean the instrument with distilled water until there are no impurities in the field of vision. Then use a 1 mL sterile syringe to push the sample slowly (injection volume: 0.5-1 mL).
Analyze the samples through supporting software (see Table of Materials). Specifically, click Start Camera and adjust the Camera level to an appropriate size (generally an intensity of 14-16 units), then select the measurement method, Standard Measurement (3 or 5 times), and click Run to collect the particles.
After collecting the particles, the computer can observe the moving sEVs particles. At the same time, set the detection threshold (generally 5) to analyze the particle size distribution so that the final counted particles are all moving sEVs rather than the background. After analyzing the particles, save and export the analysis results.
After use, wash the instrument with distilled water until there are no obvious particles in the field of vision, and turn off the laser.
NOTE: Unlike sEVs samples for TEM, sEVs samples for NTA should not be too concentrated and require larger sample volumes (at least 1 mL). Additionally, alternative methods can be used to analyze the size distribution of sEVs, such as flow cytometry and tunable resistive pulse sensing (TRPS), etc. The recommended number of particles/frames for NTA measurements (NanoSight LM10) is 40.

4. Detection of protein markers of sEVs by western blot

NOTE: According to the Minimal information for studies of extracellular vesicles (MISEV) 2018 guidelines¹⁷^,¹⁸, 5 categories of proteins are recommended for the characterization of sEVs. Evaluate at least one protein marker of each category 1 to 4 for sEVs preparation.

For the sEVs harvested in step 1.9, carefully pipette the supernatant, and add 15 µL of HEPES lysis buffer for 5 min. Then add an equal amount of loading buffer and cook in boiling water for 15 min.
In this protocol, the loading of sEVs' protein was 8-10 µg. Electrophorese the proteins in 10% gels at a constant voltage of 80 V (about 30 min).
1. When the sample enters the separating gel, adjust the voltage to 120 V (about 45 min). After the sample is 2-3 cm away from the bottom of the glass plate, disconnect the power supply and cut off the sample strip for electroporation. The composition of electrophoresis solution is described in sub-step 4.2.1.1.
  1. To prepare 5x electrophoresis solution, weigh 15.1 g, 5 g, and 94 g of Tris, SDS, and glycine, respectively, and dilute to 1 L with deionized water. When it is used, it needs to be diluted 5 times with water.
Assemble the electroporation device and put in sufficient electroporation solution (about 1 L), and electroporate the protein on the nitrocellulose (NC) membrane with a constant current of 90 mA on ice for 3.5 h.
NOTE: To prepare 10x electroporation solution, weigh 30.25 g and 144.1 g of Tris and glycine, respectively, and dilute to 1 L with deionized water. When it is used, it needs to be prepared at a ratio of 7:2:1 of deionized water: methanol: electroporation solution.
After electroporation, place the NC membrane in blocking solution (2.5 g of skimmed milk powder in 50 mL of Tris-buffered saline with 0.1% Tween 20 (TBST) for 1-2 h at RT or overnight at 4 °C. The composition of TBST is described in sub-step 4.4.1.
1. To prepare 10x TBST, weigh 60.56 g and 175.32 g of Tris and NaCl, respectively, and then add 10 mL of Tween-20 and 34 mL of concentrated hydrochloric acid. Adjust pH = 7.6 with concentrated hydrochloric acid and makeup to 2 L with deionized water. When it is used, it needs to be diluted ten times with water.
Identify the protein markers by three sEVs' markers (cluster of differentiation 9 [CD9]; β-actin; tumor susceptibility gene [TSG101]) and one non- sEVs' marker (glucose-regulated protein 94 [GRP94]).
1. Pipette 2 µL of the above antibodies and dilute at 1:500, then incubate the NC membranes at RT for 3 h or overnight at 4 °C. The diluent is the blocking solution in step 4.4.
2. After incubation with the primary antibody, wash 3 times with 1x TBST on a shaker for 10 min each time.
Place the NC membrane in the diluted secondary antibody (1:500) and shake slowly on a shaker at RT for 45-50 min. Wash it 3 times with 1x TBST on a shaker for 10 min each time.
Mix the chemiluminescent substrates A and B (see Table of Materials) at 1:1, add the mixed reagent to the NC membrane, and incubate at RT for 5 min.
Image the NC membrane using a blot imager (see Table of Materials)
1. Open the Image Lab software, and select New Experimental Protocol.
2. Select the application Blotting-colorimetric, cancel Highlight, click Run Experimental Protocol, acquire the marker, and save it.
3. Then re-select the application program Blotting-chemi, and set the Total number of images and Exposure time to 30 s and 300 s, respectively.
4. Click Run Experimental Protocol, acquire the images, and save it. Finally, remove the NC membrane and turn off the computer.
  NOTE: For step 4.6, the incubation time of the secondary antibody should not exceed 50 min; otherwise, the background will be too dark.

5. Extraction of sEVs' peptides

Wash the sEVs twice by ultracentrifugation at 110,000 × g at 4 °C for 70 min, and add 500 µL of PBS (see Table of Materials) to resuspend them, and add 5 µL of 100x protease inhibitor (see Table of Materials).
Place the sample for ultrasonic crushing (see Table of Materials). Settings for ultrasonic homogenization are as follows: power: 40 W, total time: 10 min, ultrasonic time: 3 s, and interval time: 5 s.
Centrifuge the crushed mixture at 13,700 × g for 15 min at 4 °C.
After centrifugation, add dithiothreitol (DTT; Table of Materials) to the supernatant to a final concentration of 50 mmol/L, and incubate in a water bath at 56 °C for 30 min.
Subsequently, add 50 µL of iodoacetamide (IAA; Table of Materials) to the supernatant at a concentration of 1 mol/L and leave it to react at RT for 20 min in the dark.
Centrifuge the mixture at 13,700 × g at 4 °C for 15 min, and collect the supernatant, the crude peptides extract. Store it at -20 °C for later use.
Ultrafilter the obtained peptides crude extract with a 10 kDa ultrafiltration tubes¹⁹ (see Table of Materials) to remove proteins, and centrifuge at 13,700 × g at 4 °C for 1 h. Collect the effluent and dry it in a vacuum centrifugal concentrator (see Table of Materials) at 45 °C.
Desalt the dried samples following steps 5.8.1-5.8.8. Use mass spectrometry-grade pure water as a solvent for the reagents.
1. Add 100 µL of 0.1% trifluoroacetic acid (TFA) to each sample to completely dissolve the dried peptides.
2. Add 100 µL of 100% acetonitrile (ACN) to each desalting column, centrifuge at 400 × g for 3 min at RT, and discard the effluent. Then add 100 µL of 50% ACN, centrifuge at 400 × g for 3 min, and discard the effluent.
3. Add 100 µL of 0.1% TFA to each desalting column, centrifuge at 400 × g for 3 min, and discard the effluent.
4. Repeat step 5.8.3.
5. Pipette the peptides dissolved in step 5.8.1 into the desalting column, centrifuge at 400 × g for 3 min, and recover the effluent. Repeat the sample loading step to let the peptide sample adsorb into the desalting column.
6. Repeat step 5.8.3 twice.
7. Add 50 µL of 50% ACN (containing 0.1% TFA) to the desalting column, centrifuge at 400 × g for 3 min, and collect the effluent (peptides) in a new mass spectrometry-grade centrifuge tube.

6. Drying the desalted peptides

Dry the desalted peptides in a vacuum centrifugal concentrator (Settings: V-AQ mode, temperature: 45 °C, and time: 50 min ) and use them for subsequent mass spectrometry analysis.
NOTE: The extraction process of sEVs' peptides should be performed on ice as much as possible to avoid protein degradation. All reagents used were prepared with mass spectrometry-grade water to avoid plastic contamination. For step 5.8, desalting results in the inevitable loss of the peptide samples. This loss can be reduced by repeating steps 5.8.5 and 5.8.7.

7. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis

NOTE: Analyze the peptides' sample by liquid chromatography-tandem mass spectrometry (LC-MS/MS), specifically the Orbitrap Q Exactive HF-X mass spectrometer connected with an EASY-nLC 1000 nano-high-performance LC system (see Table of Materials).

Separate the samples on a C18 analytical column (1.9 µm of grain diameter, 15 cm length x 150 µm inner diameter) at a flow rate of 60 nL/min with a 65 min gradient: 4%-15% for 4 min, 15%-28 % for 28 min, 28%-40% for 10 min, 40%-69% for 10 min, 95% constant for 7 min, 95% decreased to 6% for 1 min, and constant for 5 min (solvent A, water containing 0.1% formic acid [FA]; solvent B, 80% acetonitrile containing 0.1% FA, v/v).
Ionize the eluted peptides in a nano-electrospray ionization (nano-ESI) sprayer at 320 °C capillary temperature and spray voltage of 2.2 kV.
Collect the mass spectral data using data-dependent acquisition mode with a resolving power of 120,000 for full-scan mode and 15,000 at MS/MS mode.
1. Perform full scans in the orbitrap from scan range 250 m/z to 1800 m/z and isolation window with 1.6 m/z.
2. Select the top 20 intense ions for higher-energy collisional dissociation (HCD) fragmentation with a normalized collision energy of 29% and measure in the ion separator.
  NOTE: Typical mass spectrometric conditions were as follows: Automatic gain control (AGC) targets were 3 x 10⁶ ions for full scans and 2 x 10⁵ for MS/MS scans; the maximum injection time was 80 ms for full scans and 19 ms for MS/MS scans; and dynamic exclusion was used for 13 s.

Representative Results

For the sEVs isolated by differential ultracentrifugation (Figure 1), we evaluated their morphology, particle size distribution, and protein markers according to the International Society for Extracellular Vesicles (ISEV)¹⁷.

First, the morphology of sEVs was observed by TEM, showing a typical cup-like structure (Figure 2A). NTA showed that isolated sEVs were mostly concentrated at 136 nm (Figure 2B), which was consistent with the reported size (30-150 nm)¹. Finally, the sEVs' protein markers were identified by western blot. The results showed that isolated sEVs were significantly enriched of sEVs' markers, including CD9, β-actin, and TSG101. The endoplasmic reticulum marker, GRP94, was only detected in the whole cell lysate (Figure 2C). These results indicated that the methods employed resulted in a high level of purity for the isolated sEVs.

Figure 1: Schematic representation of isolating sEVs by differential ultracentrifugation. Carry out all centrifugations at 4 °C. Please click here to view a larger version of this figure.

Figure 2: Characterization of iBMDM-derived sEVs. (A) Isolated sEVs observed by transmission electron microscopy. Scale bar: 200 nm. (B) Particle size of the isolated sEVs by nanoparticle tracking analysis. (C) Identification of the sEVs and non- sEVs markers of sEVs by western blot. Please click here to view a larger version of this figure.

Discussion

When investigating the function of sEVs, it is imperative to attain high-purity sEVs from complex biological samples to avoid any potential contaminations. A variety of methods for sEVs isolation have been developed¹³, and among these methods, differential ultracentrifugation-based methods have shown relatively high purity of sEVs. In this study, 200 mL of cell supernatant was collected for 6 h, and about 200-300 µg of sEVs were obtained by differential ultracentrifugation. However, it should be noted that the sEVs pellet may not be visible during ultracentrifugation (step 1.8). Therefore, it is recommended to pipette the tubes’ bottom as much as possible. This step is critical and will directly affect the yield of sEVs. Additionally, further optimization of the protocol is required to improve yield, such as extending the centrifugation time or cell supernatant collection period when sEVs are precipitated at 110,000 × g (steps 1.8 and 1.9). Although the sEVs isolated by differential ultracentrifugation have high purity, it also takes a long time.

With advancements in modern mass spectrometry technology and genetic databases, tens of thousands of peptides have been identified in various organisms’ tissues and body fluids, differing significantly in source, abundance, and biological function²⁰. LC-MS/MS-based peptidomes provide a comprehensive approach to investigating the sEVs peptidome’s composition, dynamic change, and function. However, the low abundance of peptides in sEVs makes identifying sEVs peptidome unstable. Additionally, peptide extraction must be performed on ice to avoid protein degradation interfering with peptide identification. In this study, 2-4 µg of peptides required nearly 1 mg of sEVs for peptidome analysis. This requires a larger sample size than sEVs’ proteomics. At the same time, due to the extremely low peptide concentration in sEVs, more attention should be paid to plastic pollution than proteomics. Whether it is cell culture or reagent preparation, use mass spectrometry-grade consumables as much as possible. Therefore, more optimized peptide extraction methods are needed to increase the number of identified peptides in sEVs. Additionally, the peptidome database is not complete, which limits the progress of sEVs peptidome research.

Currently, most studies on sEVs focus on the proteins and microRNAs they carry, with little known about their peptide components⁴^,²¹^,²². This article provides a straightforward and easy-to-follow protocol for the study of sEVs’ peptides to further unravel sEVs’ biological function from the perspective of peptidome.

開示

The authors have nothing to disclose.

Acknowledgements

This study was supported by grants from the Natural Science Foundation of China (3157270). We thank Dr. Feng Shao (National Institute of Biological Sciences, China) for providing iBMDM.

Materials

BCA Protein Assay Kit	Beyotime Technology	P0012
CD9	Beyotime Technology	AF1192
Centrifugal filter tube	Millipore	UFC5010BK
Centrifuge bottles polypropylene	Beckman Coulter	357003	High-speed centrifuge
Chemiluminescent substrate	Thermo Fisher Scientific	34580
Dithiothreitol	Solarbio	D8220	100 g
DMEM culture medium	Cell World	N?A
GRP94	Cell Signaling Technology	20292
High-speed centrifuge	Beckman Coulter	Avanti JXN-26	Centrifuge rotor (JA-25.50)
Immortalized bone marrow-derived macrophages (iBMDM)	National Institute of Biological Sciences, China		Provided by Dr. Feng Shao (National Institute of Biological Sciences, China)
Iodoacetamide	Sigma	l1149	5 g
Microfuge tube polypropylene	Beckman Coulter	357448	1.5 mL, Tabletop ultracentrifuge
nano-high-performance LC system	Thermo Fisher Scientific	EASY-nLC 1000
Nanoparticle tracking analysis	Malvern Panalytical	NanoSight LM10	NanoSight NTA3.4
Orbitrap Q Exactive HF-X mass spectrometer	Thermo Fisher Scientific	N/A
Phosphate-buffered saline	Solarbio	P1020
Polyallomer centrifuge tubes	Beckman Coulter	326823	Ultracentrifuge
Protease inhibitor	Bimake	B14002
SpeedVac vacuum concentrator	Eppendorf	Concentrator plus
Tabletop ultracentrifuge	Beckman Coulter	Optima MAX-XP	Ultracentrifuge rotor (TLA 55)
Transmission electron microscope	HITACHI	H-7650B
TSG101	Sigma	AF8258
Ultracentrifuge	Beckman Coulter	Optima XPN-100	Ultracentrifuge rotor (SW32 Ti)
Ultrasonic cell disruptor	Scientz	SCIENTZ-IID
Western Blot imager	Bio-Rad	ChemiDocXRs	Image lab 4.0 (beta 7)
β-actin	Sigma	A3853

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Cheng, J., Zhu, J., Liu, Y., Yang, C., Zhang, Y., Liu, Y., Jin, C., Wang, J. Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages. J. Vis. Exp. (196), e65521, doi:10.3791/65521 (2023).

Identification of Peptides of Small Extracellular Vesicles from Bone Marrow-Derived Macrophages