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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. Centrifuge 12 mL of urine sample in a 15 mL conical centrifuge tube for 10 min at 2,500 x g, 4 °C to remove cell debris and large apoptotic bodies.
  2. Transfer 10 mL of the su.......

Representative Results

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.......

Discussion

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

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
1.5 mL microcentrifuge tubeLife Science ProductsM-1700C-LB
1.5 mL tube magnetic separator rackSergi Lab Supplies1005
15 mL conical centrifuge tubeCorning 352097
15 mL tube magnetic separator rackSergi Lab Supplies1002
Anti-rabbit IgG, HRP-linked AntibodyCell Signaling Technology7074P2
Benchtop incubated shakerBioerDIS-87999-3367802Bioer Thermocell Mixing Block MB-101
CD9 (D3H4P) Rabbit mAbCell Signaling Technology13403S
ChloroacetamideSigma -AldrichC0267-100GUsed for alkylation of reduced sulfide groups. Freshly prepare 400 mM in water as stock solution.
Ethyl acetate Fisher Scientific E145-4Precipitates detergents
Evosep One EvosepLiquid chromatography system
EvotipsEvosepEV2013Sample loading for Evosep One system 
EVtrapTymora AnalyticalFunctionalized magnetic beads, loading buffer, and washing buffer 
Immobilon-FL PVDF MembraneSigma -AldrichIPFL00010Blotting membrane 
NuPAGE 4-12% Bis-Tris GelInvitrogenNP0322BOXInvitrogen NuPAGE 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 12-well
NuPAGE LDS Sample Buffer (4X)InvitrogenNP0007
PBSThermoFisher10010023
Pepsep C18 15 x 75 x 1.9Bruker 1893473Separation column 
Phosphatase Inhibitor Cocktail 2Sigma -AldrichP5726-5ML100X, Phosphotase inhibitor.
Phosphatase Inhibitor Cocktail 3Sigma -AldrichP0044-1ML100X,  Phosphotase inhibitor. 
Pierce BCA Protein Assay KitThermoFisher23225
Pierce ECL Western Blotting SubstrateThermoFisher32106HRP substrate 
PolyMAC phosphopeptide enrichment kitTymora AnalyticalPolymer-based metal ion affinity capture (PolyMAC) for phosphopeptide enrichment
Sodium deoxycholate Sigma -AldrichD6750-10GDetergent for lysis buffer. Prepare 120 mM in water as stock solution.
Sodium lauroyl sarcosinate Sigma -AldrichL9150-50GDetergent for lysis buffer. Prepare 120 mM in water as stock solution.
timsTOF HTBrukerTrapped ion-mobility time-of-flight mass spectrometry
TopTip C-18 (10-200 μL) tips GlygenTT2C18.96Desalting method
TriethylamineSigma -Aldrich471283-100MLFor EV elution. 
Triethylammonium bicabonate bufferSigma -AldrichT7408-100ML1 M
Trifluoroacetic acidSigma -Aldrich302031-100ML
Tris-(2-carboxyethyl)phosphine hydrochlorideSigma -AldrichC4706Used 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 MIXThermoFisherPIA41007

References

  1. Abels, E. R., Breakefield, X. O. Introduction to extracellular vesicles: Biogenesis, RNA cargo selection, content, release, and uptake. Cell Mol Neurobiol. 36 (3), 301-312 (2016).
  2. Maacha, S., et al.

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Extracellular VesiclesBiofluidProteomicsPhosphoproteomicsEVtrapLiquid BiopsyBiomarker DiscoveryMass SpectrometryProteinPost translational ModificationPhosphorylationUrine

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