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Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins.

Ghosh A, Davey M, Chute IC, Griffiths SG, Lewis S, Chacko S, Barnett D, Crapoulet N, Fournier S, Joy A, Caissie MC, Ferguson AD, Daigle M, Meli MV, Lewis SM, Ouellette RJ - PLoS ONE (2014)

Bottom Line: We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins.All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method.Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada; Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New Brunswick, Canada.

ABSTRACT
Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

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Related in: MedlinePlus

Characterization of extracellular materials precipitated by Vn96.A. Vn96 peptides precipitate vesicular structures from conditioned cell culture media. The biotinylated-Vn96 (b-Vn96) precipitated materials from conditioned cell culture media previously incubated with the MDA-MB-231 breast cancer cell line were subjected to Proteinase K digestion. The transmission electron microscopy analysis were performed on the precipitated material from the b-Vn96 sample (left panel), proteinase K-digested b-Vn96 sample (middle panel), and the proteinase K-digested sample from b-Scr-Vn96 (right panel). The scale bars are 100 nm. B. Identification of exosome markers in the Vn96-purified EVs from conditioned cell culture media. 50 µg each of Vn96 peptide and Scr-Vn96 were incubated with 1 ml of conditioned cell culture media previously incubated with the breast cancer cell line MCF-7 at 4°C for overnight. Exosomes were also isolated from the same conditioned cell culture media by ultracentrifugation (UCF). The presence of HSP70, HSP90 and GAPDH were assessed by immunoblotting. C. CD63 immunoblot. 1 ml of pre-cleared MCF-7 conditioned cell culture media was incubated to precipitate EVs with indicated amount of peptides either overnight (O/N) at 4°C or 30 minutes at room temperature. Total cell lysate of MCF-7 (equivalent to 0.2×106 cells) was used as a positive control and conditioned cell culture media alone (c. media) was used as negative control. SDS-PAGE was performed in non-reducing conditions for CD63 immunoblots as recommended by the supplier.
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pone-0110443-g002: Characterization of extracellular materials precipitated by Vn96.A. Vn96 peptides precipitate vesicular structures from conditioned cell culture media. The biotinylated-Vn96 (b-Vn96) precipitated materials from conditioned cell culture media previously incubated with the MDA-MB-231 breast cancer cell line were subjected to Proteinase K digestion. The transmission electron microscopy analysis were performed on the precipitated material from the b-Vn96 sample (left panel), proteinase K-digested b-Vn96 sample (middle panel), and the proteinase K-digested sample from b-Scr-Vn96 (right panel). The scale bars are 100 nm. B. Identification of exosome markers in the Vn96-purified EVs from conditioned cell culture media. 50 µg each of Vn96 peptide and Scr-Vn96 were incubated with 1 ml of conditioned cell culture media previously incubated with the breast cancer cell line MCF-7 at 4°C for overnight. Exosomes were also isolated from the same conditioned cell culture media by ultracentrifugation (UCF). The presence of HSP70, HSP90 and GAPDH were assessed by immunoblotting. C. CD63 immunoblot. 1 ml of pre-cleared MCF-7 conditioned cell culture media was incubated to precipitate EVs with indicated amount of peptides either overnight (O/N) at 4°C or 30 minutes at room temperature. Total cell lysate of MCF-7 (equivalent to 0.2×106 cells) was used as a positive control and conditioned cell culture media alone (c. media) was used as negative control. SDS-PAGE was performed in non-reducing conditions for CD63 immunoblots as recommended by the supplier.

Mentions: Given the observation that Vn96 could capture HSPs that are associated with membranes, we chose to examine whether Vn96 could capture membrane-bound structures associated with extracellular HSPs from cell culture conditioned media. To generate conditioned media, the breast cancer cell line MDB-MB-231 was grown in EV-free standard cell culture media as described in the experimental procedures section and subsequently collected for downstream experiments. The conditioned growth media, as well as unused control growth media, was incubated overnight with rotation at 4°C with 100 µg/ml each of b-Vn96 or b-Scr-Vn96 peptide. Translucent precipitates were observed only in the b-Vn96 samples following centrifugation at 17,000×g. The 17,000×g pellets were washed with 1 ml of PBS three times (17,000×g) and the final pellets resuspended in 50 µl of PBS and treated with Proteinase K. TEM studies of Proteinase K-treated b-Vn96 samples revealed populations of vesicles, whereas the b-Scr-Vn96 samples showed no such structures (Figure 2A). The vesicular structures isolated using the b-Vn96 are reminiscent of previously described exosomes and EVs [40]. These results indicate the Vn96 peptide captures membrane-bound structures from cell culture growth media that are potentially EVs.


Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins.

Ghosh A, Davey M, Chute IC, Griffiths SG, Lewis S, Chacko S, Barnett D, Crapoulet N, Fournier S, Joy A, Caissie MC, Ferguson AD, Daigle M, Meli MV, Lewis SM, Ouellette RJ - PLoS ONE (2014)

Characterization of extracellular materials precipitated by Vn96.A. Vn96 peptides precipitate vesicular structures from conditioned cell culture media. The biotinylated-Vn96 (b-Vn96) precipitated materials from conditioned cell culture media previously incubated with the MDA-MB-231 breast cancer cell line were subjected to Proteinase K digestion. The transmission electron microscopy analysis were performed on the precipitated material from the b-Vn96 sample (left panel), proteinase K-digested b-Vn96 sample (middle panel), and the proteinase K-digested sample from b-Scr-Vn96 (right panel). The scale bars are 100 nm. B. Identification of exosome markers in the Vn96-purified EVs from conditioned cell culture media. 50 µg each of Vn96 peptide and Scr-Vn96 were incubated with 1 ml of conditioned cell culture media previously incubated with the breast cancer cell line MCF-7 at 4°C for overnight. Exosomes were also isolated from the same conditioned cell culture media by ultracentrifugation (UCF). The presence of HSP70, HSP90 and GAPDH were assessed by immunoblotting. C. CD63 immunoblot. 1 ml of pre-cleared MCF-7 conditioned cell culture media was incubated to precipitate EVs with indicated amount of peptides either overnight (O/N) at 4°C or 30 minutes at room temperature. Total cell lysate of MCF-7 (equivalent to 0.2×106 cells) was used as a positive control and conditioned cell culture media alone (c. media) was used as negative control. SDS-PAGE was performed in non-reducing conditions for CD63 immunoblots as recommended by the supplier.
© Copyright Policy
Related In: Results  -  Collection

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pone-0110443-g002: Characterization of extracellular materials precipitated by Vn96.A. Vn96 peptides precipitate vesicular structures from conditioned cell culture media. The biotinylated-Vn96 (b-Vn96) precipitated materials from conditioned cell culture media previously incubated with the MDA-MB-231 breast cancer cell line were subjected to Proteinase K digestion. The transmission electron microscopy analysis were performed on the precipitated material from the b-Vn96 sample (left panel), proteinase K-digested b-Vn96 sample (middle panel), and the proteinase K-digested sample from b-Scr-Vn96 (right panel). The scale bars are 100 nm. B. Identification of exosome markers in the Vn96-purified EVs from conditioned cell culture media. 50 µg each of Vn96 peptide and Scr-Vn96 were incubated with 1 ml of conditioned cell culture media previously incubated with the breast cancer cell line MCF-7 at 4°C for overnight. Exosomes were also isolated from the same conditioned cell culture media by ultracentrifugation (UCF). The presence of HSP70, HSP90 and GAPDH were assessed by immunoblotting. C. CD63 immunoblot. 1 ml of pre-cleared MCF-7 conditioned cell culture media was incubated to precipitate EVs with indicated amount of peptides either overnight (O/N) at 4°C or 30 minutes at room temperature. Total cell lysate of MCF-7 (equivalent to 0.2×106 cells) was used as a positive control and conditioned cell culture media alone (c. media) was used as negative control. SDS-PAGE was performed in non-reducing conditions for CD63 immunoblots as recommended by the supplier.
Mentions: Given the observation that Vn96 could capture HSPs that are associated with membranes, we chose to examine whether Vn96 could capture membrane-bound structures associated with extracellular HSPs from cell culture conditioned media. To generate conditioned media, the breast cancer cell line MDB-MB-231 was grown in EV-free standard cell culture media as described in the experimental procedures section and subsequently collected for downstream experiments. The conditioned growth media, as well as unused control growth media, was incubated overnight with rotation at 4°C with 100 µg/ml each of b-Vn96 or b-Scr-Vn96 peptide. Translucent precipitates were observed only in the b-Vn96 samples following centrifugation at 17,000×g. The 17,000×g pellets were washed with 1 ml of PBS three times (17,000×g) and the final pellets resuspended in 50 µl of PBS and treated with Proteinase K. TEM studies of Proteinase K-treated b-Vn96 samples revealed populations of vesicles, whereas the b-Scr-Vn96 samples showed no such structures (Figure 2A). The vesicular structures isolated using the b-Vn96 are reminiscent of previously described exosomes and EVs [40]. These results indicate the Vn96 peptide captures membrane-bound structures from cell culture growth media that are potentially EVs.

Bottom Line: We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins.All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method.Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada; Department of Chemistry and Biochemistry, Université de Moncton, Moncton, New Brunswick, Canada.

ABSTRACT
Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.

Show MeSH
Related in: MedlinePlus