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 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).</li>
	<li>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.</li>
	<li>According to the needs of the experiment, collect the cell supernatant and transfer it to 50 mL centrifuge tubes.</li>
	<li>Centrifuge the cell supernatant at 300 &#215; g for 10 min to remove the cells, discard the pellet, and transfer the supernatant to new 50 mL centrifuge tubes.</li>
	<li>Centrifuge the supernatant at 2000 &#215; 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).</li>
	<li>Centrifuge the supernatant at 10,000 &#215; 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.</li>
	<li>Centrifuge the supernatant at 110,000 &#215; g for 70 min in a swinging-bucket rotor centrifuge(see Table of Materials) to obtain crude sEV pellet.</li>
	<li>Discard the supernatant, and wash the sEVs-enriched pellet with 1 mL of PBS. Centrifuge at 110,000 &#215; 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.</li>
	<li>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.</li>
	<li>Discard the supernatant, and resuspend the pellet in 100 &#181;L of PBS, which is sEVs.</li>
	<li>Measure the total protein of sEVs using the Bicinchoninic Acid (BCA) method, following the steps below (see Table of Materials).
	<ol>
		<li>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.</li>
		<li>Add 0 &#181;L, 1 &#181;L, 2 &#181;L, 4 &#181;L, 8 &#181;L, 12 &#181;L, 16 &#181;L, and 20 &#181;L of the protein standard into the 96-well plate, and add PBS to make up 20 &#181;L. At the same time, add 18 &#181;L of HEPES lysis buffer (20 mM HEPES, 50 mM NaCl, 1 mM NaF, 0.5% Triton X-100) and 2 &#181;L of sEVs samples to the sample wells to be tested.</li>
		<li>Prepare the required BCA working solution as per the manufacturer&#39;s instructions, add 200 &#181;L of BCA working solution to each well, and place at room temperature (RT) for 20-30 min.</li>
		<li>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 &#181;L of PBS. Take 20-30 &#181;L 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 &#176;C for a short term (12 h); otherwise, they must be stored at -20 or -80 &#176;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 &#176;C for a short term (12 h).</li>
	</ol>
	</li>
</ol>
2. Observation of morphology of sEVs by transmission electron microscopy
<ol>
	<li>Place a drop of fresh sEVs sample (about 20-30 &#181;L) on the parafilm, place the number side of the copper mesh on the sEVs droplet, and let it absorb for 3 min.</li>
	<li>Absorb the excess liquid with filter paper, and then place the copper grid on a drop of distilled water and wash it twice.</li>
	<li>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.</li>
	<li>Repeat step 2.2.</li>
	<li>Place the prepared copper mesh on the sample rod and insert it into the sample stage.
	<ol>
		<li>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 (&#60;50 &#181;g/&#181;L), the adsorption time can be extended to 5 min or even longer. Alternatively, for step 1.10, a smaller volume of PBS (50 &#181;L) 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.</li>
	</ol>
	</li>
</ol>
3. Measurements of particle size distribution and concentration of sEVs by nanoparticle tracking analysis
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>For the sEVs harvested in step 1.9, carefully pipette the supernatant, and add 15 &#181;L of HEPES lysis buffer for 5 min. Then add an equal amount of loading buffer and cook in boiling water for 15 min.</li>
	<li>In this protocol, the loading of sEVs&#39; protein was 8-10 &#181;g. Electrophorese the proteins in 10% gels at a constant voltage of 80 V (about 30 min).
	<ol>
		<li>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.
		<ol>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#176;C. The composition of TBST is described in sub-step 4.4.1.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Identify the protein markers by three sEVs&#39; markers (cluster of differentiation 9 [CD9]; &#946;-actin; tumor susceptibility gene [TSG101]) and one non- sEVs&#39; marker (glucose-regulated protein 94 [GRP94]).
	<ol>
		<li>Pipette 2 &#181;L of the above antibodies and dilute at 1:500, then incubate the NC membranes at RT for 3 h or overnight at 4 &#176;C. The diluent is the blocking solution in step 4.4.</li>
		<li>After incubation with the primary antibody, wash 3 times with 1x TBST on a shaker for 10 min each time.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Mix the chemiluminescent substrates A and B (see&#160;Table of Materials) at 1:1, add the mixed reagent to the NC membrane, and incubate at RT for 5 min.</li>
	<li>Image the NC membrane using a blot imager (see Table of Materials)
	<ol>
		<li>Open the Image Lab software, and select New Experimental Protocol.</li>
		<li>Select the application Blotting-colorimetric, cancel Highlight, click Run Experimental Protocol, acquire the marker, and save it.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Extraction of sEVs&#39; peptides
<ol>
	<li>Wash the sEVs twice by ultracentrifugation at 110,000 &#215; g at 4 &#176;C for 70 min, and add 500 &#181;L of PBS (see Table of Materials) to resuspend them, and add 5 &#181;L of 100x protease inhibitor (see Table of Materials).</li>
	<li>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.</li>
	<li>Centrifuge the crushed mixture at 13,700 &#215; g for 15 min at 4 &#176;C.</li>
	<li>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 &#176;C for 30 min.</li>
	<li>Subsequently, add 50 &#181;L 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.</li>
	<li>Centrifuge the mixture at 13,700 &#215; g at 4 &#176;C for 15 min, and collect the supernatant, the crude peptides extract. Store it at -20 &#176;C for later use.</li>
	<li>Ultrafilter the obtained peptides crude extract with a 10 kDa ultrafiltration tubes19&#160;(see Table of Materials) to remove proteins, and centrifuge at 13,700 &#215; g at 4 &#176;C for 1 h. Collect the effluent and dry it in a vacuum centrifugal concentrator (see Table of Materials) at 45 &#176;C.</li>
	<li>Desalt the dried samples following steps 5.8.1-5.8.8. Use mass spectrometry-grade pure water as a solvent for the reagents.
	<ol>
		<li>Add 100 &#181;L of 0.1% trifluoroacetic acid (TFA) to each sample to completely dissolve the dried peptides.</li>
		<li>Add 100 &#181;L of 100% acetonitrile (ACN) to each desalting column, centrifuge at 400 &#215; g for 3 min at RT, and discard the effluent. Then add 100 &#181;L of 50% ACN, centrifuge at 400 &#215; g for 3 min, and discard the effluent.</li>
		<li>Add 100 &#181;L of 0.1% TFA to each desalting column, centrifuge at 400 &#215; g for 3 min, and discard the effluent.</li>
		<li>Repeat step 5.8.3.</li>
		<li>Pipette the peptides dissolved in step 5.8.1 into the desalting column, centrifuge at 400 &#215; g for 3 min, and recover the effluent. Repeat the sample loading step to let the peptide sample adsorb into the desalting column.</li>
		<li>Repeat step 5.8.3 twice.</li>
		<li>Add 50 &#181;L of 50% ACN (containing 0.1% TFA) to the desalting column, centrifuge at 400 &#215; g for 3 min, and collect the effluent (peptides) in a new mass spectrometry-grade centrifuge tube.</li>
	</ol>
	</li>
</ol>
6. Drying the desalted peptides
<ol>
	<li>Dry the desalted peptides in a vacuum centrifugal concentrator (Settings: V-AQ mode, temperature: 45 &#176;C, and time: 50 min ) and use them for subsequent mass spectrometry analysis. 
	NOTE: The extraction process of sEVs&#39; 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.</li>
</ol>
7. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
NOTE: Analyze the peptides&#39; 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).
<ol>
	<li>Separate the samples on a C18 analytical column (1.9 &#181;m of grain diameter, 15 cm length x 150 &#181;m 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).</li>
	<li>Ionize the eluted peptides in a nano-electrospray ionization (nano-ESI) sprayer at 320 &#176;C capillary temperature and spray voltage of 2.2 kV.</li>
	<li>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.
	<ol>
		<li>Perform full scans in the orbitrap from scan range 250 m/z to 1800 m/z and isolation window with 1.6 m/z.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

Isolating Small Extracellular Vesicles and Extracting Their Peptidome

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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.

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

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

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

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 ...

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.

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Research

JoVE Journal

Biochemistry

1.2K Views.  Anhui Medical University. 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 antimicrobi...

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

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5&#39;-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5&#39;-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5&#39;-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5&#39;-H2PO4-(8-oxoG)G CUA AAA GU], [5&#39;-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5&#39;-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

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 ...

Characterization of Cell Wall-Related Enzymes in RWA-Infested Wheat Cultivars

Elucidating &#946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as &#946;-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and &#946;-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 &#176;C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. &#946;-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using &#946;-1,3-glucan and &#946;-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 &#176;C, and thermal stability between 30 &#176;C and 70 &#176;C. &#946;-1,3-glucanase substrate specificity was determined at 25 &#176;C and 40 &#176;C using curdlan and barley &#946;-1,3-1,4-glucan substrates. Additionally, the &#946;-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and &#946;-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

Author Spotlight: Integrating Biochemical Functions of &#946;-Glucanases and Peroxidase Enzymes in Wheat-RWA Interaction

The present protocol describes procedures used to study and characterize cell wall-related enzymes, mainly &#946;-1,3-glucanase and peroxidase, in wheat plants. Their activity levels increase during wheat-RWA interaction and are involved in the plant defense response through cell wall reinforcement, which deters aphid feeding.

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of ...

 Peptide Hydrogels: Methods for Environmentally Responsive Gelation of ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...