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 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.
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.
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
2. EV isolation using the EVtrap approach
3. Characterization of EVs by western blotting
4. Sample preparation for proteomics and phosphoproteomics analysis
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.
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 > 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).
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 (> 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 (< 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 µg 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.
The authors have nothing to disclose.
This work has been funded in part by NIH grants 3RF1AG064250 and R44CA239845.
1.5 mL microcentrifuge tube | Life Science Products | M-1700C-LB | |
1.5 mL tube magnetic separator rack | Sergi Lab Supplies | 1005 | |
15 mL conical centrifuge tube | Corning | 352097 | |
15 mL tube magnetic separator rack | Sergi Lab Supplies | 1002 | |
Anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology | 7074P2 | |
Benchtop incubated shaker | Bioer | DIS-87999-3367802 | Bioer Thermocell Mixing Block MB-101 |
CD9 (D3H4P) Rabbit mAb | Cell Signaling Technology | 13403S | |
Chloroacetamide | Sigma -Aldrich | C0267-100G | Used for alkylation of reduced sulfide groups. Freshly prepare 400 mM in water as stock solution. |
Ethyl acetate | Fisher Scientific | E145-4 | Precipitates detergents |
Evosep One | Evosep | Liquid chromatography system | |
Evotips | Evosep | EV2013 | Sample loading for Evosep One system |
EVtrap | Tymora Analytical | Functionalized magnetic beads, loading buffer, and washing buffer | |
Immobilon-FL PVDF Membrane | Sigma -Aldrich | IPFL00010 | Blotting membrane |
NuPAGE 4-12% Bis-Tris Gel | Invitrogen | NP0322BOX | Invitrogen NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well |
NuPAGE LDS Sample Buffer (4X) | Invitrogen | NP0007 | |
PBS | ThermoFisher | 10010023 | |
Pepsep C18 15 x 75 x 1.9 | Bruker | 1893473 | Separation column |
Phosphatase Inhibitor Cocktail 2 | Sigma -Aldrich | P5726-5ML | 100X, Phosphotase inhibitor. |
Phosphatase Inhibitor Cocktail 3 | Sigma -Aldrich | P0044-1ML | 100X, Phosphotase inhibitor. |
Pierce BCA Protein Assay Kit | ThermoFisher | 23225 | |
Pierce ECL Western Blotting Substrate | ThermoFisher | 32106 | HRP substrate |
PolyMAC phosphopeptide enrichment kit | Tymora Analytical | Polymer-based metal ion affinity capture (PolyMAC) for phosphopeptide enrichment | |
Sodium deoxycholate | Sigma -Aldrich | D6750-10G | Detergent for lysis buffer. Prepare 120 mM in water as stock solution. |
Sodium lauroyl sarcosinate | Sigma -Aldrich | L9150-50G | Detergent for lysis buffer. Prepare 120 mM in water as stock solution. |
timsTOF HT | Bruker | Trapped ion-mobility time-of-flight mass spectrometry | |
TopTip C-18 (10-200 μL) tips | Glygen | TT2C18.96 | Desalting method |
Triethylamine | Sigma -Aldrich | 471283-100ML | For EV elution. |
Triethylammonium bicabonate buffer | Sigma -Aldrich | T7408-100ML | 1 M |
Trifluoroacetic acid | Sigma -Aldrich | 302031-100ML | |
Tris-(2-carboxyethyl)phosphine hydrochloride | Sigma -Aldrich | C4706 | 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°C (avoid multiple freeze-thaw cycles). |
Trypsin/Lys-C MIX | ThermoFisher | PIA41007 |