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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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Comparative miRNA-seq data for Vn96- and UCF-purified EVs from conditioned cell culture media.A. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from the indicated conditioned cell culture media using either ultracentrifugation or the Vn96 peptide. For example, MCF7_UCF and MCF7_VN96 indicate that EVs were purified from conditioned cell culture media previously incubated with MCF-7 cells by ultracentrifugation and the Vn96 peptide, respectively. High Pearson correlations between ultracentrifugation and Vn96 peptide methods of EV purification from the same sample validate Vn96 as an EV purification tool. B. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from MCF-7 versus MDA-MB-231 conditioned cell culture media using the same purification method. C. Venn diagram of miRNAs contained in EVs isolated from MCF-7 conditioned cell culture media using different methods (ultracentrifugation, Vn96 peptide and a commercially-available exosome purification kit). Less than 10% differences were observed in the miRNA populations between the ultracentrifugation and Vn96 peptide methods, and the commercial kit and Vn96 peptide methods (left panel), but a wider variation in miRNA populations was observed in EVs from different cell lines (right panel).
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pone-0110443-g004: Comparative miRNA-seq data for Vn96- and UCF-purified EVs from conditioned cell culture media.A. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from the indicated conditioned cell culture media using either ultracentrifugation or the Vn96 peptide. For example, MCF7_UCF and MCF7_VN96 indicate that EVs were purified from conditioned cell culture media previously incubated with MCF-7 cells by ultracentrifugation and the Vn96 peptide, respectively. High Pearson correlations between ultracentrifugation and Vn96 peptide methods of EV purification from the same sample validate Vn96 as an EV purification tool. B. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from MCF-7 versus MDA-MB-231 conditioned cell culture media using the same purification method. C. Venn diagram of miRNAs contained in EVs isolated from MCF-7 conditioned cell culture media using different methods (ultracentrifugation, Vn96 peptide and a commercially-available exosome purification kit). Less than 10% differences were observed in the miRNA populations between the ultracentrifugation and Vn96 peptide methods, and the commercial kit and Vn96 peptide methods (left panel), but a wider variation in miRNA populations was observed in EVs from different cell lines (right panel).

Mentions: Comparative assessments of miRNA extracted from EVs isolated using Vn96 and the UCF method for one cell line-type revealed very similar profiles with high Pearson correlations, minimal expression variation and less than 5% population variability (Figure 4A). On the other hand, higher dispersion, differential expression and high population variability were observed when miRNA cargos of UCF-purified EVs were profiled for two different cell lines (Figure 4B). Similar wide variations were also observed in the miRNA profiles of EVs precipitated from the same two cell lines using the Vn96 peptide. Furthermore, populations of differentially-expressed miRNAs identified in the EVs of the two cell lines were highly similar irrespective of the isolation method used (UCF or Vn96) as shown in the normalized heat map in Text S4.


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)

Comparative miRNA-seq data for Vn96- and UCF-purified EVs from conditioned cell culture media.A. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from the indicated conditioned cell culture media using either ultracentrifugation or the Vn96 peptide. For example, MCF7_UCF and MCF7_VN96 indicate that EVs were purified from conditioned cell culture media previously incubated with MCF-7 cells by ultracentrifugation and the Vn96 peptide, respectively. High Pearson correlations between ultracentrifugation and Vn96 peptide methods of EV purification from the same sample validate Vn96 as an EV purification tool. B. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from MCF-7 versus MDA-MB-231 conditioned cell culture media using the same purification method. C. Venn diagram of miRNAs contained in EVs isolated from MCF-7 conditioned cell culture media using different methods (ultracentrifugation, Vn96 peptide and a commercially-available exosome purification kit). Less than 10% differences were observed in the miRNA populations between the ultracentrifugation and Vn96 peptide methods, and the commercial kit and Vn96 peptide methods (left panel), but a wider variation in miRNA populations was observed in EVs from different cell lines (right panel).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4201556&req=5

pone-0110443-g004: Comparative miRNA-seq data for Vn96- and UCF-purified EVs from conditioned cell culture media.A. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from the indicated conditioned cell culture media using either ultracentrifugation or the Vn96 peptide. For example, MCF7_UCF and MCF7_VN96 indicate that EVs were purified from conditioned cell culture media previously incubated with MCF-7 cells by ultracentrifugation and the Vn96 peptide, respectively. High Pearson correlations between ultracentrifugation and Vn96 peptide methods of EV purification from the same sample validate Vn96 as an EV purification tool. B. Scatter plot comparing normalized expression profiles of miRNAs contained in EVs isolated from MCF-7 versus MDA-MB-231 conditioned cell culture media using the same purification method. C. Venn diagram of miRNAs contained in EVs isolated from MCF-7 conditioned cell culture media using different methods (ultracentrifugation, Vn96 peptide and a commercially-available exosome purification kit). Less than 10% differences were observed in the miRNA populations between the ultracentrifugation and Vn96 peptide methods, and the commercial kit and Vn96 peptide methods (left panel), but a wider variation in miRNA populations was observed in EVs from different cell lines (right panel).
Mentions: Comparative assessments of miRNA extracted from EVs isolated using Vn96 and the UCF method for one cell line-type revealed very similar profiles with high Pearson correlations, minimal expression variation and less than 5% population variability (Figure 4A). On the other hand, higher dispersion, differential expression and high population variability were observed when miRNA cargos of UCF-purified EVs were profiled for two different cell lines (Figure 4B). Similar wide variations were also observed in the miRNA profiles of EVs precipitated from the same two cell lines using the Vn96 peptide. Furthermore, populations of differentially-expressed miRNAs identified in the EVs of the two cell lines were highly similar irrespective of the isolation method used (UCF or Vn96) as shown in the normalized heat map in Text S4.

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