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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 ide....
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
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.......
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
This work has been funded in part by NIH grants 3RF1AG064250 and R44CA239845.
....Name | Company | Catalog Number | Comments |
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 |
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