Proteomics

Dataset Information

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Isolation and characterization of extracellular vesicles from tissues


ABSTRACT: Extracellular vesicles (EVs) are lipid bi-layered membrane structures released by all cells. Most EV studies have been performed using cell lines or body fluids, and studies on tissue-derived EVs are limited. Here we present a protocol to isolate EV subpopulations directly from tissues. This approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations were characterized by electron microscopy and RNA profiling. Additionally, their protein cargo was determined with mass spectrometry, Western blot, and ExoView™. The protocol requires extra steps compared to EV isolation from cell culture medium. Tissue-EV isolation can be performed in 22 hours, but a simplified version can be completed in 8 hours. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanoma Cell

SUBMITTER: Proteomics Core Facility  

LAB HEAD: Cecilia Lässer

PROVIDER: PXD021694 | Pride | 2020-11-25

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
2969_ProteomeDiscoverer22.msf Msf
2969_ProteomeDiscoverer22.pdResult Other
Fusion_181018_05.raw Raw
Fusion_181018_06.raw Raw
Fusion_181018_07.raw Raw
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Publications

Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.

Crescitelli Rossella R   Lässer Cecilia C   Jang Su Chul SC   Cvjetkovic Aleksander A   Malmhäll Carina C   Karimi Nasibeh N   Höög Johanna L JL   Johansson Iva I   Johansson Iva I   Fuchs Johannes J   Thorsell Annika A   Gho Yong Song YS   Olofsson Bagge R R   Lötvall Jan J  

Journal of extracellular vesicles 20200211 1


The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs <i>in vivo</i>. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small  ...[more]

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