Characterization of Protein Content Present in Exosomes Isolated from Conditioned Media and Urine

Ankit Sinha1, Javier Alfaro1, Thomas Kislinger1

1 Princess Margaret Cancer Centre, Toronto, Ontario
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.9
DOI:  10.1002/cpps.23
Online Posting Date:  February, 2017
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Cells secrete biomolecules into the extracellular space as a way of intercellular communication. Secreted proteins can act as ligands that engage specific receptors—on the same cell, nearby cells, or distant cells—and induce defined signaling pathways. Proteins and other biomolecules can also be packaged as cargo molecules within vesicles that are released to the extracellular space (termed extracellular vesicles or EVs). A subclass of such EVs, exosomes have been shown to horizontally transfer information. In recent years, exosomes have sparked tremendous interest in biological research, both for the discovery of novel biomarkers and for the identification of signaling molecules, as part of their cargo. Although multiple methods have been described for the isolation of exosomes, described here is a simple differential centrifugation approach that is well suited for the isolation of exosomes from conditioned cell culture media and urine. Mass spectrometry provides an ideal method to comprehensively analyze the protein cargo of exosomes. © 2017 by John Wiley & Sons, Inc.

Keywords: exosome isolation; proteomics; differential centrifugation; secretome

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation and Proteomic Characterization of Exosomes from Serum‐Free Secretomes
  • Alternate Protocol 1: Isolation and Proteomic Characterization of Exosome from Cells Cultured in the Presence of Serum
  • Basic Protocol 2: Isolation and Proteomic Characterization of Exosomes Isolated from Expressed Prostatic Secretions in Urine (EPS‐Urine)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation and Proteomic Characterization of Exosomes from Serum‐Free Secretomes

  • Phosphate buffered saline (PBS), pH 7.4 ( appendix 2E)
  • Complete medium: appropriate tissue culture media containing 100 U/ml penicillin–streptomycin and 2 mM L‐glutamine
  • Secretome medium: appropriate tissue culture media (without phenol red or fetal bovine serum)
  • Denaturation buffer: 50% (v/v) 2,2,2‐trifluoroethanol in PBS, pH 7.4
  • 500 mM dithiothreitol (DTT) in water
  • 1 M iodoacetamide in water (prepared fresh)
  • 100 mM ammonium bicarbonate buffer, pH 8.0 (prepared fresh)
  • 2 M CaCl 2 in water
  • 1 μg/μl trypsin (sequencing grade) in 50 mM acetic acid; alternatively Lys‐C/trypsin mixtures (Promega, cat. no. V5071) could be used
  • Trifluoroacetic acid
  • Priming solution: 80% acetonitrile and 0.1% formic acid in water
  • Washing solution: 0.1% formic acid in water
  • Elution solution: 60% acetonitrile and 0.1% formic acid in water
  • Buffer A: 0.1% MS formic acid in MS‐grade water
  • Buffer B: 0.1% MS formic acid in MS‐grade acetonitrile
  • 15‐cm tissue culture dishes
  • 15‐ml centrifugal filters with 3 kDa MWCO (EMD Millipore)
  • 10‐ml polycarbonate ultracentrifuge tube (Beckman Coulter, cat. no. 355603)
  • Desalting tips: OMIX C18 pipette tips (Agilent, USA)
  • Heat block set at 60°C
  • Refrigerated centrifuge capable of reaching 300, 2000, and 10,000 × g (e.g., Eppendorf 5810R)
  • Ultracentrifuge capable of 120,000 × g (e.g., Beckman Coulter Type 70.1 Ti)
  • SpeedVac (Thermo Fisher Scientific)
  • Easy Spray ES803 50‐cm C18 reverse‐phase column (Thermo Fisher Scientific) or similar
  • QExactive mass spectrometer (Thermo Fisher Scientific) coupled to an EasyLC 1200 nano‐liquid chromatography system (Thermo Fisher Scientific) or similar
  • Additional reagents and equipment for determination of protein concentration (unit 3.4; Olson and Markwell, ) and microvolume spectrophotometry (unit 3.10; Desjardins et al., )

Alternate Protocol 1: Isolation and Proteomic Characterization of Exosome from Cells Cultured in the Presence of Serum

  Materials (also see protocol 1)
  • Sterile fetal bovine serum (Invitrogen)
  • 0.22‐μm vacuum filter (Gibco)

Basic Protocol 2: Isolation and Proteomic Characterization of Exosomes Isolated from Expressed Prostatic Secretions in Urine (EPS‐Urine)

  Materials (also see protocol 1)
  • EPS‐urine samples (obtained from the BioBank under an ethics approved protocol)
  • Resuspension buffer: 10 mM triethanolamine (pH 7.6), 250 mM sucrose
  • Dithiothreitol
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Literature Cited

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