Isolation of Exosomes for the Purpose of Protein Cargo Analysis with the Use of Mass Spectrometry

  • Monika Pietrowska
  • Sonja Funk
  • Marta Gawin
  • Łukasz Marczak
  • Agata Abramowicz
  • Piotr Widłak
  • Theresa Whiteside
Part of the Methods in Molecular Biology book series (MIMB, volume 1654)


Exosomes are intercellular messengers with a high potential for diagnostic and therapeutic utility. It is believed that exosomes present in body fluids are responsible for providing signals which inhibit immune cells, interfere with antitumor immunity, and thus influence the response to treatment and its effect. One of the most interesting issues in exosome studies is proper addressing of their cargo composed of nucleic acids and proteins. Effective and selective isolation of extracellular vesicles and identification of proteins present in exosomes has turned out to be a challenging aspect of their exploration. Here we propose a novel approach that is based on isolation of exosomes by mini-size-exclusion chromatography which allows efficient, rapid, and reliable isolation of morphologically intact and functionally active exosomes without the need of ultracentrifugation. The purpose of this chapter is to describe a simple and high-throughput method to isolate, purify, and identify exosomal proteins using a mass spectrometry approach. The proposed protocol compiles the expertise of two research groups specialized in exosome research and in mass spectrometry-based proteomics. The protocol combines differential centrifugation followed by ultrafiltration, centrifugation-based filtration, and gel filtration on Sepharose 2B in order to obtain exosomal fractions characterized by only low contamination with albumin.

Key words

Albumin removal Cell culture Exosomes Filter-aided sample preparation Mass spectrometry Peptide assay Proteomics Size exclusion chromatography Ultrafiltration 



This work was supported by the National Science Centre, Poland, Grant 2013/11/B/NZ7/01512.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Monika Pietrowska
    • 1
  • Sonja Funk
    • 2
    • 3
  • Marta Gawin
    • 1
  • Łukasz Marczak
    • 4
  • Agata Abramowicz
    • 1
  • Piotr Widłak
    • 1
  • Theresa Whiteside
    • 2
  1. 1.Center for Translational Research and Molecular Biology of CancerMaria Sklodowska—Curie Memorial Cancer Center and Institute of Oncology, Gliwice BranchGliwicePoland
  2. 2.Department of PathologyUniversity of PittsburghPittsburghUSA
  3. 3.Department of OtolaryngologyUniversity of Duisburg-EssenEssenGermany
  4. 4.Institute of Bioorganic ChemistryPolish Academy of SciencesPoznańPoland

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