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.

1. Isolation of small extracellular vesicles
NOTE: Perform all centrifugation in steps 1.1-1.11 at 4 &#176;C.
<ol>
	<li>Preparation of sEVs-free fetal bovine serum (FBS): Centrifuge FBS overnight at 110,000 &#215; g at 4 &#176;C through an ultracentrifuge (see Table of Materials) to remove endogenous sEVs. Collect the supernatant, filter sterilize it with a 0.2 &#181;m ultrafiltration membrane, and store it at -20 &#176;C.</li>
	<li>Plate about 3 x ...

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

<ol>
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W., Zhu, H. J., Takahashi, N., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+isolation+and+characterization:+toward+clinical+application.">Extracellular vesicle isolation and characterization: toward clinical application.</a> The Journal of Clinical Investigation. 126 (4), 1152-1162 (2016).</li><li>Li, P., Kaslan, M., Lee, S. H., Yao, J., Gao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+exosome+isolation+techniques.">Progress in exosome isolation techniques.</a> Theranostics. 7 (3), 789-804 (2017).</li><li>Palanski, B. A., 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=An+efficient+urine+peptidomics+workflow+identifies+chemically+defined+dietary+gluten+peptides+from+patients+with+celiac+disease.">An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease.</a> Nature Communications. 13, 888 (2022).</li><li>Kalaora, S., 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=Identification+of+bacteria-derived+HLA-bound+peptides+in+melanoma.">Identification of bacteria-derived HLA-bound peptides in melanoma.</a> Nature. 592 (7852), 138-143 (2021).</li><li>Hamley, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+bioactive+peptides+for+biomaterials+design+and+therapeutics.">Small bioactive peptides for biomaterials design and therapeutics.</a> Chemical Reviews. 117 (24), 14015-14041 (2017).</li><li>Lotvall, J., 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=Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions:+a+position+statement+from+the+International+Society+for+Extracellular+Vesicles.">Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.</a> Journal of Extracellular Vesicles. 3, 26913 (2014).</li><li>Thery, C., 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=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.">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.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Kim, Y. G., Lone, A. M., Saghatelian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+proteolysis+of+bioactive+peptides+using+a+peptidomics+approach.">Analysis of the proteolysis of bioactive peptides using a peptidomics approach.</a> Nature Protocols. 8 (9), 1730-1742 (2013).</li><li>Lyapina, I., Ivanov, V., Fesenko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidome:+Chaos+or+inevitability.">Peptidome: Chaos or inevitability.</a> International Journal of Molecular Sciences. 22 (23), 13128 (2021).</li><li>Keller, M. D., 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=Decoy+exosomes+provide+protection+against+bacterial+toxins.">Decoy exosomes provide protection against bacterial toxins.</a> Nature. 579 (7798), 260-264 (2020).</li><li>Koeppen, K., 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=Let-7b-5p+in+vesicles+secreted+by+human+airway+cells+reduces+biofilm+formation+and+increases+antibiotic+sensitivity+of+P.+aeruginosa.">Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (28), e2105370118 (2021).</li></ol>

The authors declare that they have no competing financial interests.

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 &#181;g of sEVs were obtained by differential ultracentrifugation. However, it should...

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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Beyotime Technology</td>
			<td>P0012</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9</td>
			<td>Beyotime Technology</td>
			<td>AF1192</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal filter tube</td>
			<td>Millipore</td>
			<td>UFC5010BK</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles polypropylene</td>
			<td>Beckman Coulter</td>
			<td>357003</td>
			<td>High-speed centrifuge</td>
		</tr>
		<tr>
			<td>Chemiluminescent substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>34580</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Solarbio</td>
			<td>D8220</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>DMEM culture medium</td>
			<td>Cell World</td>
			<td>N?A</td>
			<td></td>
		</tr>
		<tr>
			<td>GRP94</td>
			<td>Cell Signaling Technology</td>
			<td>20292</td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Avanti JXN-26</td>
			<td>Centrifuge rotor (JA-25.50)</td>
		</tr>
		<tr>
			<td>Immortalized bone marrow-derived macrophages (iBMDM)</td>
			<td>National Institute of Biological Sciences, China</td>
			<td></td>
			<td>Provided by Dr. Feng Shao (National Institute of Biological Sciences, China)</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>l1149</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Microfuge tube polypropylene</td>
			<td>Beckman Coulter</td>
			<td>357448</td>
			<td>1.5 mL, Tabletop ultracentrifuge&#160;</td>
		</tr>
		<tr>
			<td>nano-high-performance LC system</td>
			<td>Thermo Fisher Scientific</td>
			<td>EASY-nLC 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoparticle tracking analysis&#160;</td>
			<td>Malvern Panalytical</td>
			<td>NanoSight LM10</td>
			<td>NanoSight NTA3.4</td>
		</tr>
		<tr>
			<td>Orbitrap Q Exactive HF-X mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyallomer centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td>Ultracentrifuge</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Bimake</td>
			<td>B14002</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac vacuum concentrator</td>
			<td>Eppendorf</td>
			<td>Concentrator plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima MAX-XP</td>
			<td>Ultracentrifuge rotor (TLA 55)</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>HITACHI</td>
			<td>H-7650B</td>
			<td></td>
		</tr>
		<tr>
			<td>TSG101</td>
			<td>Sigma</td>
			<td>AF8258</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima XPN-100</td>
			<td>Ultracentrifuge rotor (SW32 Ti)</td>
		</tr>
		<tr>
			<td>Ultrasonic cell disruptor</td>
			<td>Scientz</td>
			<td>SCIENTZ-IID</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blot imager</td>
			<td>Bio-Rad</td>
			<td>ChemiDocXRs</td>
			<td>Image lab 4.0 (beta 7)</td>
		</tr>
		<tr>
			<td>&#946;-actin</td>
			<td>Sigma</td>
			<td>A3853</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of peptides of small extracellular vesicles from bone marrow-derived macrophages

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of &lt;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.

Author Spotlight: Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages 

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

We explore the functional roles of extracellular vesicles in innate immunity. We have developed a protocol to isolate small extracellular vesicles, or sEVs, via differential ultracentrifugation and identify the peptidome by LC-MS/MS, revealing the key biological functions of the peptides in sEVs. The extracellular vesicles have been shown to mediate intercellular communication by transporting cargo to recipient cells, such as proteins, lipids, and nucleic acids.By releasing antimicrobial components, they act the first line of defense against invading microbes in innate immunity. While differential ultracentrifugation yields highly pure small extracellular sEVs, it can be time-consuming and often results in low yields. In our study, these are major challenges for the stable identification of low-abundance peptides in the sEVs.

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 (F...

Watch this Scientific Journal Video about Peptidome Extraction from Small Extracellular Vesicles Isolated from Bone Marrow-Derived Macrophages at JoVE.com

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

Small extracellular vesicles (sEVs) are typically secreted by the exocytosis of multivesicular bodies (MVBs). These nanovesicles with a diameter of ...

identification-peptides-small-extracellular-vesicles-from-bone-marrow

Research

JoVE Journal

Biochemistry

Isolating Small Extracellular Vesicles (sEVs) by Differential Ultracentrifugation

Isolating Small Extracellular Vesicles (SEVs) by Differential Ultracentrifugation  

To begin, centrifuge FBS overnight in an ultracentrifuge at 110, 000 G and four degrees Celsius to remove the endogenous sEVs. Sterilize the supernatant by filtering it through a 0.2 micron ultrafiltration membrane to yield sEVs-free FBS. Next in a 150 millimeter culture dish, plate about 3 times 10 to the seventh immortalized bone-marrow-derived macrophages in 20 milliliters of DMEM culture medium.Incubate the dish at 37 degrees Celsius under 5%carbon dioxide overnight prior to collecting the sEVs from the macrophages. The next day, after discarding the medium, wash the cells with phosphate-buffered saline or PBS. Replace the medium with DMEM containing 10%sEVs-free free fetal bovine serum before incubating the cells at 37 degrees Celsius and 5%carbon dioxide.Based on the experiment's requirements, collect and transfer the cells'supernatant to 50 milliliter centrifuge tubes. Centrifuge the tubes at 300 G for 10 minutes to remove the cells. Transfer the result in supernatant from each tube to a new 50 milliliter centrifuge tube and discard the pellet.This time, centrifuge the supernatant at 2000 G for 10 minutes to remove dead cells. Then transfer the supernatant to new high-speed centrifuge tubes and discard the pellets. Next to remove debris and microvesicles, centrifuge the obtained supernatant at 10, 000 G for 30 minutes using a high-speed centrifuge.Transfer the resulting supernatant to new ultracentrifuge tubes by adding 35 milliliters in each tube and discarding the pellets. Centrifuge the ultracentrifuge tubes in a swinging bucket rotor centrifuge at 110, 000 G for 70 minutes before discarding the supernatant. Wash the resulting crude sEVs-enriched pellet with one milliliter of PBS.Again, add one milliliter of PBS to the washed pellet in one of the tubes, and mix by pipetting continuously. Transfer the one milliliter of resultant PBS suspension to another tube containing the pellet, and repeat the same until all pellets in the tubes are mixed by pipetting. Transfer the resultant one milliliter of sEVs-rich PBS to a new ultracentrifuge tube.Then centrifuge the new tube in a tabletop ultracentrifuge at 110, 000 G for 70 minutes. After discarding the supernatant, wash the resulting crude sEVs-enriched pellet with 100 microliters of PBS. Add 1.2 milliliters of protein preparation solution in a standard protein tube to dissolve the 30 milligrams of BSA present in it.Dilute the resulting protein standard solution with PBS to reduce its concentration from 25 to 0.5 milligrams per milliliter. Add eight different volumes of the diluted protein standard solution into wells of a 96-well plate, and bring the volume to 20 microliters in each well using PBB. Add 18 microliters of HEPES lysis buffer to the sample wells, followed by the addition of two microliters of the sEVs samples.Next, add 200 microliters of BCA working solution prepared per the manufacturer's instructions into each well. Allow the plate to rest at room temperature for 20 to 30 minutes. Finally, measure the absorbance at 562 nanometers using a microplate reader.Calculate the total protein content in the sEVs using the obtained results. The transmission electron microscopy images of the isolated sEVs displayed a typical cup-like morphology. Nanoparticle tracking analysis showed that the isolated sEVs were mostly concentrated at 136 nanometers.Western blot showed that isolated sEVs were significantly enriched of sEVs markers, including CD9, beta-actin, and TSG101. The endoplasmic reticulate marker GRP94 was only detected in the whole-cell lysate. The results indicated that the method employed yielded sEVs with a high level of purity.

Extraction of Peptides from Isolated Small Extracellular Vesicles (sEVs)

To begin, wash the isolated small extracellular vesicles or SEVs twice by ultracentrifugation at 110, 000 G and four degrees Celsius for 70 minutes. Re-suspend the vesicles in 500 microliters of phosphate buffered saline or PBS. Then, add five microliters of 100X protease inhibitor to it.Subject the sample to ultrasonic crushing at 40 watts for 10 minutes, with three second ultrasonic time, and five second interval time. Centrifuge the crushed mixture at 13, 700 G for 15 minutes at four degrees Celsius. To the obtained supernatant, add dithiothreitol to a final concentration of 50 millimolar.And incubate it in a water bath at 56 degrees Celsius for 30 minutes. Then add 50 microliters of one molar iodoacetamide to the supernatant. And allow it to react at room temperature for 20 minutes in the dark.Centrifuge the mixture at 13, 700 G and four degrees Celsius for 15 minutes. And transfer the supernatant containing the crude peptide extract to a 10 kilodalton ultracentrifuge tube. Remove protein from the crude peptide by centrifuging the ultra filtration tube at 13, 700 G and four degrees Celsius for one hour.Dry the collected effluent in a vacuum centrifugal concentrator at 45 degrees Celsius. For desalting, use mass spectrometry grade pure water as a solvent for all reagents. First, dissolve the dry peptides in 100 microliters of 0.1%trifluoracetic acid or TFA, and keep it aside.Then add 100 microliters of pure acetonitrile to each desalting column. Centrifuge the columns at 400 G for three minutes at room temperature, and discard the effluent. Then add 100 microliters of 50%acetonitrile per desalting column, and repeat the centrifugation as mentioned previously before discarding the effluent.Next, add 100 microliters of 0.1%TFA to each desalting column before centrifuging as mentioned previously. Once again, discard the effluent. Now, pipet the desalt peptides into the desalting column.Centrifuge the columns as mentioned previously to load the sample. Add the recovered effluent back to the desalting column to repeat the loading. Then, add 100 microliters of 0.1%TFA.Centrifuge as mentioned previously, and discard the effluent. Finally, add 50 microliters of 50%acetonitrile containing 0.1%TFA to the peptide in the desalting column. After centrifusion, as mentioned previously, collect the effluent in a new mass spectrometry grade centrifuge tube.

1.1K Views.  Anhui Medical University.  This protocol describes a procedure to isolate small extracellular vesicles from macrophages by differential ultracentrifugation and extract the peptidome for identification by mass spectrometry.

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

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