This work has been funded in part by NIH grants 3RF1AG064250 and R44CA239845.

All urine samples were collected from healthy individuals after informed consent. The experiments were compliant with all ethical standards involving human samples and conform to the guidelines from Purdue University Human Research Protection Program.
1. Sample collection
<ol>
	<li>Centrifuge 12 mL of urine sample in a 15 mL conical centrifuge tube for 10 min at 2,500 x g, 4 &#176;C to remove cell debris and large apoptotic bodies.</li>
	<li>Transfer 10 mL of the supernatant into a new 15 mL tube and proceed with EV isolation. 
	&#8203;NOTE: The protocol can be paused here, and the samples can be stored at -80 &#176;C and thawed at 37 &#176;C upon use.</li>
</ol>
2. EV isolation using the EVtrap approach
<ol>
	<li>Add 0.5 mL of loading buffer (1:10 v/v ratio) and 100 &#181;L of EVtrap bead slurry (1:50 v/v ratio) to the sample according to the manufacturer&#39;s instructions.</li>
	<li>Incubate the sample by end-over-end rotation for 30 min at room temperature.</li>
	<li>Pellet the sample by placing the 15 mL conical tube on a magnetic separator rack. Remove the supernatant.</li>
	<li>Resuspend the beads in 1 mL of washing buffer and transfer the suspension to a 1.5 mL microcentrifuge tube. Gently pipette to resuspend the EV-bound beads.</li>
	<li>Place the tube on a 1.5 mL microcentrifuge tube magnetic separator rack and aspirate the supernatant. Use a P200 pipette to completely aspirate the supernatant and avoid aspirating the beads.</li>
	<li>Wash the beads with 1 mL of washing buffer. Add the buffer immediately after aspirating the supernatant to prevent the beads from drying out.</li>
	<li>Wash the beads 2x with 1 mL of phosphate-buffered saline (PBS) at room temperature.</li>
	<li>Incubate the beads with 100 &#181;L of freshly prepared 100 mM triethylamine for 10 min at room temperature and collect the eluted solution containing EVs using a 1.5 mL microcentrifuge tube magnetic separator rack. 
	NOTE: To prepare 1 mL of 100 mM triethylamine, dilute 14 &#181;L of triethylamine solution in water.</li>
	<li>Repeat Step 2.8 and combine the eluted solutions. Dry the eluate using a vacuum centrifuge concentrator at 4 &#176;C. 
	NOTE: The experiment can be paused here. Dried EV samples can be stored at -80 &#176;C for several months without adverse effects on the following steps or the results.</li>
</ol>
3. Characterization of EVs by western blotting
<ol>
	<li>Resuspend 5% of the dried EV sample (equivalent to 0.5 mL of the urine sample) in 20 &#181;L of 1x lithium dodecyl sulfate (LDS) sample buffer with 10 mM dithiothreitol (DTT). 
	NOTE: EVs from 0.5 mL of urine are enough for detecting CD9 (an EV marker24) signal in western blotting.</li>
	<li>Boil the sample for 5 min at 95 &#176;C. Load the sample on a polyacrylamide gel and perform electrophoresis and immunoblotting following standard protocols25.</li>
	<li>After transferring the proteins onto a low-fluorescence polyvinylidene fluoride (PVDF) membrane, block the membrane with 1% bovine serum albumin (BSA) in tris-buffered saline tween-20 (TBST) for 1 h at room temperature. To prepare 1 L of TBST buffer, add 19 mM Tris base, 137 mM NaCl, and 1 mL of Tween-20; adjust pH to 7.4 using HCl.</li>
	<li>Incubate membrane with rabbit anti-CD9 antibody at 1:5,000 ratio in 1% BSA in TBST at 4 &#176;C overnight or for 2 h at room temperature.</li>
	<li>Wash the membrane 3x for 5 min each with TBST. Incubate the membrane with anti-rabbit IgG, HRP-linked secondary antibody at 1:5000 ratio in 1% BSA in TBST for 1 h at room temperature.</li>
	<li>Wash the membrane 3x for 5 min each with TBST. Add the commercially obtained enhanced chemiluminescence (ECL) substrates (1:1 ratio) onto the membrane and detect signals on a chemiluminescence imaging system.</li>
</ol>
4. Sample preparation for proteomics and phosphoproteomics analysis
<ol>
	<li>Prepare fresh lysis buffer containing 12 mM sodium deoxycholate (SDC), 12 mM sodium lauroyl sarcosinate (SLS), 100 mM triethylammonium bicarbonate buffer (TEAB), 10 mM tris-(2-carboxyethyl)phosphine (TCEP), 40 mM chloroacetamide (CAA), and 1x phosphatase inhibitor cocktails. 
	NOTE: The stock solutions are listed in the description of the Table of Materials. The lysis buffer is prepared by adding the stock solutions to achieve the desired concentrations depending on the required volume.</li>
	<li>Solubilize the dried EV sample in 100 &#181;L of lysis buffer and heat the sample for 10 min at 95 &#176;C with shaking at 1,100 rpm.</li>
	<li>After cooling the sample to room temperature, dilute it five-fold by adding 400 &#181;L of 50 mM TEAB.</li>
	<li>Measure the protein concentration using a BCA assay kit as per the manufacturer&#39;s instructions. Use the lysis buffer diluted five-fold with 50 mM TEAB as blank.</li>
	<li>Add trypsin/Lys-C mix at 1:50 w/w enzyme-to-protein ratio and incubate the sample at 37 &#176;C overnight with shaking at 1,100 rpm.</li>
	<li>Add 50 &#181;L of 10% trifluoroacetic acid (TFA) to acidify the sample.</li>
	<li>Add 600 &#181;L of ethyl acetate to the samples and vortex the mixture for 2 min.</li>
	<li>Centrifuge the sample for 3 min at 20,000 x g and remove the upper layer (organic layer). Avoid disturbing the interface during aspiration.</li>
	<li>Repeat steps 4.7-4.8. Dry the aqueous phase using a vacuum centrifuge concentrator.</li>
	<li>Resuspend the dried sample in 200 &#181;L of 0.1% TFA to acidify peptides and desalt the sample using a C18 desalting tip according to the manufacturer&#39;s instructions. Condition the tip with 200 &#181;L of 0.1% TFA in 80% acetonitrile, followed by 2x with 200 &#181;L of 0.1% TFA. Load the acidified peptide sample into the tip and then wash the tip 3x with 200 &#181;L of 0.1% TFA. Elute the peptides with 200 &#181;L of 0.1% TFA in 80% acetonitrile.</li>
	<li>Dry the eluate using a vacuum centrifuge concentrator. Dry 2% of the peptide sample (equivalent to 0.2 mL of the urine sample) separately for proteomics analysis. Use the rest (98%) of the sample for phosphoproteomics analysis.</li>
	<li>Enrich phosphopeptides from the sample using a phosphopeptide enrichment kit according to the manufacturer&#39;s instructions. Perform the steps described below for enrichment.
	<ol>
		<li>Resuspend the dried sample in 200 &#181;L of loading buffer. Add 50 &#181;L of the beads to the sample and shake vigorously for 20 min at room temperature. Load the sample with the beads to the fritted tip and centrifuge for 1 min at 100 x g.</li>
		<li>Wash the tip with 200 &#181;L of loading buffer, followed by washing buffer 1, then washing buffer 2. Perform all three washing steps by centrifuging once for 2 min at 20 x g and once for 1 min at 100 x g.</li>
		<li>Put the tip with beads into a new tube to collect the eluted phosphopeptides. Add 50 &#181;L of elution buffer to the tip and centrifuge once for 2 min at 20 x g. Add another 50 &#181;L of elution buffer to the tip and centrifuge once for 2 min at 20 x g. Centrifuge one final time for 1 min at 100 x g.</li>
	</ol>
	</li>
	<li>Dry the eluted phosphopeptides using a vacuum centrifuge concentrator.</li>
</ol>
5. LC-MS/MS analysis
NOTE: Different LC-MS/MS systems/settings and data-acquisition methods, such as data-dependent acquisition (DDA) can be used.
<ol>
	<li>Resuspend the dried proteome/phosphoproteome samples in 0.1% formic acid (solvent A) and load the samples according to the manufacturer&#39;s instructions.</li>
	<li>Inject the samples into trapped ion-mobility time-of-flight MS through the liquid chromatography (LC) system and use the preset standardized Whisper 40 samples per day method. Peptides are separated on a 15 cm C18 column (75 &#181;m inner diameter, 1.9 &#181;m particle size) as mentioned in the Table of Materials.</li>
	<li>For proteomics analysis, acquire data using a parallel accumulation-serial fragmentation combined with data-independent acquisition (dia-PASEF) acquisition method with a mass range per ramp spanning from 300-1200 m/z and from 0.6-1.50 1/K0 with a cycle time of 1.38 s.</li>
	<li>For phosphoproteomics analysis, acquire data using a dia-PASEF acquisition method with a mass range per ramp spanning from 400-1550 m/z and 0.6-1.50 1/K0 with a cycle time of 1.38 s.</li>
	<li>Load the raw files into proteomics software and perform signal extraction, identification, and quantitation using a library-free data independent acquisition workflow.
	<ol>
		<li>For search settings, use Homo sapiens database, specific digest types with trypsin/P enzymes, 7 minimal peptide length, 52 maximum peptide length, two missed cleavage, carbamidomethyl at cysteine as fixed modification, acetyl protein N-term, oxidation at methionine, and phosphorylation at serine, threonine, and tyrosine (for phosphoproteomics analysis) as variable modifications, and 5 as maximum variable modifications. Set the FDR at PSM, peptide, and protein group to 0.01. 
		NOTE: Spectronaut software was used in this protocol. Other DIA data searching software such as DIA-NN and PEAKS are also commonly used. If data was acquired in DDA mode, software such as MaxQuant and Proteome Discoverer are applicable.</li>
	</ol>
	</li>
</ol>

Enhancing EV Isolation for Precise Biofluid Analysis

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The authors declare a competing financial interest. Anton Iliuk and W. Andy Tao are co-founders of Tymora Analytical Operations, which developed EVtrap beads and commercialized PolyMAC phosphopeptide enrichment kit.

Effective EV isolation is an essential prerequisite to detecting low-abundant proteins and phosphoproteins in EVs. Despite the development of numerous methods to fulfill this need, the majority still suffer from limitations such as poor recovery or low reproducibility, which impede their utilization in large-scale studies and routine clinical settings. DUC is generally considered as the most common method for EV isolation, and the additional washing steps are normally applied to help increase the purity of target EVs<sup...

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles secreted by all types of cells and are present in biofluids such as blood, urine, saliva, etc.1,2,3,4. EVs carry a cargo of diverse bioactive molecules which reflect the physiological and pathological state of their host cells and, therefore function as crucial factors in disease progression4,5,6. Moreover, extensive studies have established that EV-based disease markers can be identified prior to the onset of symptoms or the physiological detection of tumors5,6,7.
Phosphorylation acts as a key mechanism in cellular signaling and regulation. Therefore, phosphoproteins provide a valuable source for biomarker discovery as aberrant phosphorylation events are associated with dysregulated cellular signaling pathways and metastatic disease development such as cancer8,9,10. Although profiling phosphorylation dynamics allows for the identification of disease-specific phosphoprotein signatures as potential biomarkers, the low abundance and dynamic nature of phosphoproteins pose major challenges in developing phosphoproteins as biomarkers11,12. Notably, the low-abundant phosphoproteins encapsulated within EVs are protected from external enzymatic digestion in the extracellular environment8. Consequently, EVs and EV-derived phosphoproteins offer an ideal source for biomarker discovery in the early-stage detection of cancer and other diseases.
Although analysis of protein phosphorylation in EVs offers a valuable resource for understanding cancer signaling and early-stage disease diagnosis, the lack of efficient EV isolation methods presents a major barrier. EV isolation is commonly achieved through differential ultracentrifugation (DUC)13. However, this method is time-consuming and is not suitable for clinical implications due to low throughput and poor reproducibility13,14. Alternative EV isolation approaches, such as polymer-induced precipitation15, are limited by low specificity due to co-precipitation of non-EV proteins. Affinity-based approaches, including antibody-based affinity capture16 and affinity filtration17, offer enhanced specificity but are restricted to a relatively low recovery rate due to small volume.
To address the issues in exploring phosphoprotein dynamics in EVs, our group has developed extracellular vesicles total recovery and purification (EVtrap) technique based on chemical affinity to capture EVs onto functionalized magnetic beads18. Previous results have demonstrated that this magnetic bead-based EV isolation method is highly effective in isolating EVs from a wide range of biofluid samples and is able to achieve much higher EV yield while minimizing contamination compared to DUC and other existing isolation methods18,19. We have successfully utilized EVtrap and a titanium-based phosphopeptide enrichment method developed by our group20 to profile the phosphoproteome of EVs derived from diverse biofluids and to detect potential phosphoprotein biomarkers for various diseases19,21,22.
Here, we present a protocol based on EVtrap for the isolation of circulating EVs. The protocol focuses on the urinary EVs. We also demonstrate the characterization of isolated EVs using western blotting. We then detail the sample preparation and mass spectrometry (MS) acquisition for both proteomics and phosphoproteomics analyses. This protocol provides an efficient and reproducible workflow for profiling the urinary EV proteome and phosphoproteome, which will facilitate further studies on EVs and their clinical applications23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Life Science Products</td>
			<td>M-1700C-LB</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube magnetic separator rack</td>
			<td>Sergi Lab Supplies</td>
			<td>1005</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tube</td>
			<td>Corning&#160;</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube magnetic separator rack</td>
			<td>Sergi Lab Supplies</td>
			<td>1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074P2</td>
			<td></td>
		</tr>
		<tr>
			<td>Benchtop incubated shaker</td>
			<td>Bioer</td>
			<td>DIS-87999-3367802</td>
			<td>Bioer Thermocell Mixing Block MB-101</td>
		</tr>
		<tr>
			<td>CD9 (D3H4P) Rabbit mAb</td>
			<td>Cell Signaling Technology</td>
			<td>13403S</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroacetamide</td>
			<td>Sigma -Aldrich</td>
			<td>C0267-100G</td>
			<td>Used for alkylation of reduced sulfide groups. Freshly prepare 400 mM in water as stock solution.</td>
		</tr>
		<tr>
			<td>Ethyl acetate&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>E145-4</td>
			<td>Precipitates detergents</td>
		</tr>
		<tr>
			<td>Evosep One&#160;</td>
			<td>Evosep</td>
			<td></td>
			<td>Liquid chromatography system</td>
		</tr>
		<tr>
			<td>Evotips</td>
			<td>Evosep</td>
			<td>EV2013</td>
			<td>Sample loading for Evosep One system&#160;</td>
		</tr>
		<tr>
			<td>EVtrap</td>
			<td>Tymora Analytical</td>
			<td></td>
			<td>Functionalized magnetic beads, loading buffer, and washing buffer&#160;</td>
		</tr>
		<tr>
			<td>Immobilon-FL PVDF Membrane</td>
			<td>Sigma -Aldrich</td>
			<td>IPFL00010</td>
			<td>Blotting membrane&#160;</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Gel</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>Invitrogen NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4X)</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsep C18 15 x 75 x 1.9</td>
			<td>Bruker&#160;</td>
			<td>1893473</td>
			<td>Separation column&#160;</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail 2</td>
			<td>Sigma -Aldrich</td>
			<td>P5726-5ML</td>
			<td>100X, Phosphotase inhibitor.</td>
		</tr>
		<tr>
			<td>Phosphatase Inhibitor Cocktail 3</td>
			<td>Sigma -Aldrich</td>
			<td>P0044-1ML</td>
			<td>100X,&#160; Phosphotase inhibitor.&#160;</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce ECL Western Blotting Substrate</td>
			<td>ThermoFisher</td>
			<td>32106</td>
			<td>HRP substrate&#160;</td>
		</tr>
		<tr>
			<td>PolyMAC phosphopeptide enrichment kit</td>
			<td>Tymora Analytical</td>
			<td></td>
			<td>Polymer-based metal ion affinity capture (PolyMAC) for phosphopeptide enrichment</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate&#160;</td>
			<td>Sigma -Aldrich</td>
			<td>D6750-10G</td>
			<td>Detergent for lysis buffer. Prepare 120 mM in water as stock solution.</td>
		</tr>
		<tr>
			<td>Sodium lauroyl sarcosinate&#160;</td>
			<td>Sigma -Aldrich</td>
			<td>L9150-50G</td>
			<td>Detergent for lysis buffer. Prepare 120 mM in water as stock solution.</td>
		</tr>
		<tr>
			<td>timsTOF HT</td>
			<td>Bruker</td>
			<td></td>
			<td>Trapped ion-mobility time-of-flight mass spectrometry</td>
		</tr>
		<tr>
			<td>TopTip C-18 (10-200 &#956;L) tips&#160;</td>
			<td>Glygen</td>
			<td>TT2C18.96</td>
			<td>Desalting method</td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma -Aldrich</td>
			<td>471283-100ML</td>
			<td>For EV elution.&#160;</td>
		</tr>
		<tr>
			<td>Triethylammonium bicabonate buffer</td>
			<td>Sigma -Aldrich</td>
			<td>T7408-100ML</td>
			<td>1 M</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma -Aldrich</td>
			<td>302031-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Sigma -Aldrich</td>
			<td>C4706</td>
			<td>Used for reducion of disulfide bonds. Prepare 200 mM in water as stock solution. Aliquot the stock solution into small volume and store it in at-20&#176;C (avoid multiple freeze-thaw cycles).</td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C MIX</td>
			<td>ThermoFisher</td>
			<td>PIA41007</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

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

This protocol demonstrates a comprehensive workflow from the isolation of EVs to downstream proteomics and phosphoproteomics analyses (Figure 1). The triplicate urine samples were subjected to EV isolation. The isolated EVs were characterized by western blotting and subsequently processed for mass spectrometry-based proteomics sample preparation including protein extraction, enzymatic digestion, and peptide cleanup. For phosphoproteomics analysis, the phosphopeptides were further enriched ba...

Watch this Scientific Journal Video about Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery at JoVE.com

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 ...

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JoVE Journal

Biochemistry

2.1K Views.  Purdue University. 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.