Isolation of Exosomes for the Purpose of Protein Cargo Analysis with the Use of Mass Spectrometry
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 wordsAlbumin 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.
- 1.Théry C, Clayton A, Amigorena S et al (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol 30:3.22.1–3.22.29Google Scholar
- 11.Filter Aided Sample preparation (FASP) Method. http://www.biochem.mpg.de/226356/FASP. Accessed 7 Jun 2016
- 12.Tutorials. http://www.biochem.mpg.de/226455/Tutorials. Accessed 7 Jun 2016