Name: chemical affinity-based isolation of extracellular vesicles from biofluids for proteomics and phosphoproteomics analysis
Uploaded: 2023-10-27T13:10:00.000+00:00
Duration: 9 min 28 s
Description: Watch this Scientific Journal Video about Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery at JoVE.com

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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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=Generating+high+quality+libraries+for+DIA+MS+with+empirically+corrected+peptide+predictions.">Generating high quality libraries for DIA MS with empirically corrected peptide predictions.</a> Nat Commun. 11 (1), 1548 (2020).</li><li>Skowronek, P., 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=Rapid+and+in-depth+coverage+of+the+(Phospho-)proteome+with+deep+libraries+and+optimal+window+design+for+dia-PASEF.">Rapid and in-depth coverage of the (Phospho-)proteome with deep libraries and optimal window design for dia-PASEF.</a> Mol Cell Proteomics. 21 (9), 100279 (2022).</li></ol>

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 EVs27,28. This procedure leads to a more tedious and time-consuming DUC process (&#62; 6 h). Moreover, low EV recovery yield after DUC has been reported by multiple studies,29,30 and shown in the results in Figure 2. In comparison, EVtrap offers efficient EV isolation (&#60; 1 h) and a high recovery yield (Figure 2). Notably, the successful application of this workflow marks a crucial advancement for the identification of significant EV markers for various diseases and cancers19,21,22 However, EVtrap is incapable of isolating specific subpopulations of EVs, such as microvesicles or exosomes, as it captures the entire EV population.
In this protocol, we used 2% and 98% of the peptide sample for proteomics and phosphoproteomics analyses. Since the EVs isolated from 10 mL of urine by EVtrap typically yield 100-200 &#181;g of total proteins, 98% of the peptides after digestion and cleanup is sufficient for the phosphopeptide enrichment. If a labeling step, such as tandem mass tag (TMT) labeling, is required for quantitative MS analysis, peptide concentration measurement following the desalting step can be performed to normalize the peptide amount and improve the accuracy of quantification31.
While this protocol primarily highlights the use of EVtrap for isolating urinary EVs, it is noteworthy to mention that the versatility of this approach has been demonstrated in processing various sample types32,33,34. Based on previous results, the condition used in this protocol was applicable to isolate EVs from cell culture media. For the isolation of cell-secreted EVs, the cells are cultivated under fetal bovine serum (FBS)-free or EV-depleted FBS conditions to prevent co-isolation of excessive FBS-derived EVs with cell-secreted EVs, which will undermine the reliability of results35. Additionally, the isolated EVs from other sample types can be characterized by western blotting analysis using several common EV markers, including CD9, CD63, CD81, tumor susceptibility gene 101 protein (TSG101), and ALG-2-interacting protein X (ALIX)36.
To increase coverage and improve quantification in MS analysis, building a project-specific library for searching DIA data is an alternative option. Due to the complexity of the spectra produced in the DIA mode, a spectral library containing a collection of reference spectra is beneficial for increasing identification confidence and improving coverage. High-quality spectral libraries can be generated by performing DDA analysis on the same samples or pre-fractionated samples37. The library can also be used to determine optimal windows for dia-PASEF and further improve identification38.
Taken together, the presented protocol provides a simple and effective method for isolating EVs from urine samples for proteomics and phosphoproteomics analyses. By implementing this protocol, we expect improved detection of low-abundant EV proteins and phosphoproteins as biomarkers for early-stage disease detection and longitudinal monitoring. Furthermore, researchers have the great opportunity to advance the field of EV research and contribute to better diagnostic and therapeutic strategies.

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><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&amp;nbsp;</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&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>E145-4</td><td>Precipitates detergents</td></tr><tr><td>Evosep One&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>EVtrap</td><td>Tymora Analytical</td><td></td><td>Functionalized magnetic beads, loading buffer, and washing buffer&amp;nbsp;</td></tr><tr><td>Immobilon-FL PVDF Membrane</td><td>Sigma -Aldrich</td><td>IPFL00010</td><td>Blotting membrane&amp;nbsp;</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&amp;nbsp;</td><td>1893473</td><td>Separation column&amp;nbsp;</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,&amp;nbsp; Phosphotase inhibitor.&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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 &amp;mu;L) tips&amp;nbsp;</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.&amp;nbsp;</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&amp;deg;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.

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 based on metal ion-functionalized soluble nanopolymers. Both peptide and phosphopeptide samples were analyzed by high-resolution ion mobility mass spectrometry under data-independent mode. The result raw files were searched against Homo sapiens database, and library-free data-independent acquisition workflow was performed for identification and MS2-level quantification.
To characterize the isolated EVs and estimate the recovery yield, we first loaded an equivalent of 0.5 mL of urine containing EVs onto the gel for western blotting to detect the EV marker CD9 (Figure 2A). In addition, we included the EVs isolated from the same volume of urine using the most used method for EV isolation, differential ultracentrifugation (DUC), for comparison. The results showed that EVtrap was able to produce much higher CD9 signals compared to DUC, indicating an effective capture of EVs by the beads. Further quantitative values for each CD9 band signal demonstrated that EVtrap achieved a recovery yield of ~99% compared to the 5-fold intensity of the direct urine control, while DUC only recovered ~1.5% of EVs (Figure 2B).
By loading 2% of each sample onto the LC-MS/MS for proteomic profiling, we identified &#62; 11,000 unique peptides from ~2,200 unique proteins (Figure 3A), indicating that this workflow provides an in-depth coverage of EV proteome. A high degree of overlap in protein identifications across samples was observed, with 72% of unique proteins being consistently identified in all three replicates (Figure 3B). Moreover, we compared the identification results with the ExoCarta database (Figure 3C)26. Notably, out of the top 100 EV markers and proteins, we successfully identified ~90 of these EV proteins, suggesting an unbiased and complete profiling of EV proteins through this proteomics analysis. To assess the quantitative precision, we further evaluated the distribution of coefficients of variation (CV) for the protein quantification results (Figure 3D). A low medium CV (5.7%) indicates the high reproducibility and reliability of the procedure, including EV isolation, sample preparation, and MS detection.
For the phosphoproteomics analysis, we used 98% of each peptide sample for the phosphopeptide enrichment and identified ~800 unique phosphopeptides corresponding to ~350 unique phosphoproteins (Figure 4A). The enrichment yielded an average of 72% phosphoserine (pS) peptides, 22% phosphothreonine (pT), and 6% phosphotyrosine (pY) peptides, respectively (Figure 4B). In terms of the identification reproducibility of the three replicates, 42% of phosphopeptides were identified by all three analyses, and there was a ~50% overlap between every two analyses (Figure 4C). A medium CV of 21.8% was determined for the quantification of phosphopeptides, suggesting an acceptable quantitative reproducibility using this protocol (Figure 4D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65844/65844fig01.jpg" /> 
Figure 1: Schematic workflow used for isolation of urinary extracellular vesicle (EV) and downstream analyses. EVs are isolated from urine samples through the extracellular vesicles total recovery and purification (EVtrap) approach, and the isolated EVs are directly subjected to western blotting analysis. For LC-MS/MS analysis, proteins are extracted from EVs and digested into peptides. The peptide samples after the cleanup steps can be used for proteomics analysis or phosphoproteomics analysis after phosphopeptide enrichment. Both proteomics and phosphoproteomics samples are analyzed by LC-MS/MS. Identification and quantification are then performed using data independent acquisition (DIA) workflow method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65844/65844fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65844/65844fig02.jpg" /> 
Figure 2: Characterization of isolated EVs by western blotting. (A) Detection of EV marker CD9 from 0.1 mL of direct urine sample (n=1), EVs isolated using differential centrifugation (DUC) approach (n=3), and EVs isolated using EVtrap approach (n=3). (B) Quantification of western blotting signals in (A) is presented as the percentage recovery relative to the direct urine sample (5-fold intensity = 100%). For the DUC and EVtrap samples, the bar plot displays the average intensities and standard deviations (represented by error bars) from the triplicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65844/65844fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65844/65844fig03.jpg" /> 
Figure 3: Proteomics analysis of isolated EVs. (A) The total number of identified unique proteins and peptides in triplicates. (B) Venn diagram showing the overlap in identified unique proteins between replicates. (C) The number of identified unique proteins corresponding to the top 100 EV markers in the ExoCarta database. (D) Density plot showing the distribution of coefficient of variation (CV) for protein quantification. The median CV value is highlighted with a red dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65844/65844fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65844/65844fig04.jpg" /> 
Figure 4: Phosphoproteomics analysis of isolated EVs. (A) The total number of identified unique phosphoproteins and phosphopeptides in triplicates. (B) Percentage composition of pSTY peptides after phosphopeptide enrichment. (C) Venn diagram showing the overlap in identified unique phosphopeptides between replicates. (D) Density plot showing the distribution of CV for phosphopeptide quantification. The median CV value is highlighted with a red dashed line. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65844/65844fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

chemical-affinity-based-isolation-extracellular-vesicles-from

Nanyang Technological University

Research

JoVE Journal

Biochemistry

2.8K Views.  Purdue University. We werken aan de ontdekking van extracellulaire biomarkers op basis van blaasjes. Een belangrijke stap is om ze efficiënt en selectief te isoleren. EVtrap, dat is gebaseerd op scheiding op basis van magnetische kralen, kan ons echt helpen door een hoge doorvoer te bieden, gemakkelijk te gebruiken, in het bijzonder gemakkelijk voor klinische toepassingen.We hebben dit protocol gebruikt voor het ontdekken van eiwitten en de fosfoproteïne-biomarkers voor verschillende ziekten zoals bors...

Video: Auteur in de schijnwerpers: EVtrap bevorderen voor High-Throughput Proteomics bij de ontdekking van biomarkers voor ziekten 

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

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 ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Immunology and Infection

Characterization of Immune Cell-derived Extracellular Vesicles and Studying Functional Impact on Cell Environment

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident immune cells in the brain, and sometimes the infiltrating bone marrow-derived macrophages (BMDMs), regulate the inflammatory profile of their microenvironment in the CNS. It is now accepted that the extracellular vesicle (EV) populations from immune cells act as immune mediators. Thus, their collection and isolation are important to identify their contents but also evaluate their biological effects on recipient cells. The present data highlight chronological requirements for EV isolation from microglia cells or blood macrophages including the ultracentrifugation and size-exclusion chromatography (SEC) steps. A non-targeted proteomic analysis permitted the validation of protein signatures as EV markers and characterized the biologically active EV contents. Microglia-derived EVs were also functionally used on primary culture of neurons to assess their importance as immune mediators in the neurite outgrowth. The results showed that microglia-derived EVs contribute to facilitate the neurite outgrowth in vitro. In parallel, blood macrophage-derived EVs were functionally used as immune mediators in spheroid cultures of C6 glioma cells, the results showing that these EVs control the glioma cell invasion in vitro. This report highlights the possibility to evaluate the EV-mediated immune cell functions but also understand the molecular bases of such a communication. This deciphering could promote the use of natural vesicles and/or the in vitro preparation of therapeutic vesicles in order to mimic immune properties in the microenvironment of CNS pathologies.

The present report highlights chronological requirements for extracellular vesicle (EV) isolation from microglia or blood macrophages. Microglia-derived EVs were evaluated as regulators of the neurite outgrowth while blood macrophage-derived EVs were studied in the control of C6 glioma cell invasion in in vitro assays. The goal is to better understand these EV functions as immune mediators in specific microenvironments.

The neuroinflammatory state of the central nervous system (CNS) plays a key role in physiological and pathological conditions. Microglia, the resident ...

Neuroscience

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

Bioengineering

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Biology

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Cardiovascular Biomarker Studies Using Plasma Extracellular Vesicle Proteome

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research due to their role in regulating intercellular communication, potentially serving as valuable biomarkers for cardiovascular disease. However, the EV proteome and its potential as a biomarker in cardiovascular diagnostics remain poorly understood. This protocol presents a standardized method for the isolation and quantification of plasma-derived EV and the analysis of their protein cargo using plasma samples from patients presenting to the Chest Pain Unit of a large university hospital. Following routine phlebotomy, EV are isolated from plasma by differential ultracentrifugation. The enrichment of specific EV marker proteins in EV isolates is visualized by immunoblotting, and average size distribution and plasma EV concentrations are quantified by nanoparticle tracking analysis. Finally, ultra-performance liquid chromatography-tandem mass spectrometry is employed for label-free analysis of the EV proteome. This protocol thus provides a comprehensive approach to study and use plasma-derived EV as potential carriers of critical biological information as well as to explore their potential as novel biomarkers.

Author Spotlight: Advancing the Analysis of Plasma Extracellular Vesicle Proteome for Cardiovascular Biomarker Studies

This article describes a methodology for the isolation, characterization, and quantification of human plasma-derived extracellular vesicles (EV) and presents a workflow for label-free analysis of the EV proteome using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

Medicine

Efficient Recycling of Polycarbonate Ultracentrifuge Tubes for Proteomics

Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ultracentrifuge (UC) tubes are used to endure the associated high centrifugal forces. EV proteomics is an advancing field and validated re-use protocols for these tubes are lacking. Re-using consumables for low-yield protein isolation protocols and downstream proteomics requires reagent compatibility with mass spectroscopy acquisitions, such as the absence of centrifuge tube-derived synthetic polymer contamination, and sufficient removal of residual proteins.
This protocol describes and validates a method for cleaning polycarbonate UC tubes for re-use in EV proteomics experiments. The cleaning process involves immediate submersion of UC tubes in H2O to prevent protein drying, washing in 0.1% sodium dodecyl sulfate (SDS) detergent, rinsing in hot tap water, demineralized water, and 70% ethanol. To validate the UC tube re-use protocol for downstream EV proteomics, used tubes were obtained following an experiment isolating EVs from cardiovascular tissue using differential UC and density gradient separation. Tubes were cleaned and the experimental process was repeated without EV samples comparing blank never-used UC tubes to cleaned UC tubes. The pseudo-EV pellets obtained from the isolation procedures were lysed and prepared for liquid chromatography-tandem mass spectrometry using a commercial protein sample preparation kit with modifications for low-abundance protein samples.
Following cleaning, the number of identified proteins was reduced by 98% in the pseudo-pellet versus the previous EV isolation sample from the same tube. Comparing a cleaned tube against a blank tube, both samples contained a very small number of proteins (&#8804;20) with 86% similarity. The absence of polymer peaks in the chromatograms of the cleaned tubes was confirmed. Ultimately, the validation of a UC tube cleaning protocol suitable for the enrichment of EVs will reduce the waste produced by EV laboratories and lower the experimental costs.

Author Spotlight: Effective Reuse of Polycarbonate Tubes for Extracellular Vesicle Isolation

A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments.

Single-use laboratory plastics exacerbate the pollution crisis and contribute to consumable costs. In extracellular vesicle (EV) isolation, polycarbonate ...

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

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