Rapid Isolation of Extracellular Vesicles from Cell Culture and Biological Fluids Using a Synthetic Peptide with Specific Affinity for Heat Shock Proteins

Ghosh, Anirban; Davey, Michelle; Chute, Ian C.; Griffiths, Steven G.; Lewis, Scott A.; Chacko, Simi; Barnett, David A.; Crapoulet, Nicolas; Fournier, Sébastien; Joy, Andrew P.; Caissie, Michelle C.; Ferguson, Amanda D.; Daigle, Mélissa; Meli, Maria‐Victoria; Lewis, Stephen M.; Ouellette, Rodney J.

doi:10.1371/journal.pone.0110443

Cited by 157 publications

(152 citation statements)

References 49 publications

(40 reference statements)

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“…Nanoparticle tracking analysis (NTA) and electron microscopy (EM) have been performed on Vn96 mediated extracellular vesicle isolations of MCF-10A and MCF-7 cell culture media previously [7,26]. Both studies found particle size distributions consistent with small EVs.…”

Section: Discussionmentioning

confidence: 99%

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Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

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Section: Discussionmentioning

confidence: 99%

“…We have recently developed an EV capture method that uses a synthetic peptide called Vn96, which has binding affinity for heat shock proteins that are expressed in high abundance on the surface of EVs[7]. Vn96 is a small (27-mer) peptide, with an amino acid sequence of PSQGKGRGLSLSFRSWGALTLGEFLKL and a molecular weight of 2.9 kDa.…”

Section: Introductionmentioning

confidence: 99%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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“…Now, there are 3 confirmed ways to import exogenous proteins into exosomes [84–88]. The first way is to use the transfection technique to load the exogenous proteins into exosomes directly [85, 86].…”

Section: Immunotherapy Strategymentioning

confidence: 99%

Tumor-Related Exosomes Contribute to Tumor-Promoting Microenvironment: An Immunological Perspective

Chen

Jiang

Xia

et al. 2017

Journal of Immunology Research

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Exosomes are a kind of cell-released membrane-form structures which contain proteins, lipids, and nucleic acids. These vesicular organelles play a key role in intercellular communication. Numerous experiments demonstrated that tumor-related exosomes (TEXs) can induce immune surveillance in the microenvironment in vivo and in vitro. They can interfere with the maturation of DC cells, impair NK cell activation, induce myeloid-derived suppressor cells, and educate macrophages into protumor phenotype. They can also selectively induce effector T cell apoptosis via Fas/FasL interaction and enhance regulatory T cell proliferation and function by releasing TGF-β. In this review, we focus on the TEX-induced immunosuppression and microenvironment change. Based on the truth that TEXs play crucial roles in suppressing the immune system, studies on modification of exosomes as immunotherapy strategies will also be discussed.

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“…The glass substrates were further annealed at 560°C in order to modify the morphology from the multilayers of gold nanoparticles aggregates to nano-islands. In order to capture the exosomes, the affinity based synthetic polypeptide called Venceremin, Vn96 is used [6]. This molecule is specifically designed and validated to capture exosomes, having a strong affinity towards the canonical heat shock proteins (HSP), present on the surface of exosomes.…”

Section: Extended Abstractmentioning

confidence: 99%

Gold Nano-Island Platform for the Microfluidic Plasmonic Detection of Exosomes

Bathini¹,

Raju²,

Bădilescu³

et al. 2018

International Conference of Theoretical and Applied Nanoscience and Nanotechnology

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Extended AbstractNoble metals (gold and silver) possess interesting plasmonic properties, allowing the detection at the nano-scale is by observing their LSPR band shift, due to binding events and change of the refractive index [1][2]. A shift towards the longer wavelength of the local LSPR band is noticed upon the binding of bio-molecules onto the metal nanoparticles immobilized on a substrate. While a great number of publications exist on the LSPR phenomenon and its applications, there are only a few trying to integrate LSPR biosensors and microfluidics [3][4]. Recently microfluidics-based designs have been developed and implemented by several researchers in order to isolate and quantify biomolecules which are bio markers for the early diagnosis of cancer. Exosomes are the carriers of genetical and molecular information from parent to distal cells [5]. At present, the commonly used protocols for the isolation and detections of exosomes are the high-speed centrifugation and ultracentrifugation which are time consuming, with relatively low yield. Therefore, a versatile and efficient technique is required to for the isolation and detections of exosomes for further diagnosis at clinical level.The objective of the present work is the plasmonic detection of exosomes based on the sensitivity of the Au LSPR band to binding events. As mentioned earlier, the sensing mechanism makes use of the change in the position of the Au LSPR band due to the binding of the chemical entities and biomolecules to the immobilized gold nano-island, resulting in a shift of the band towards longer wavelengths. For the capture and detection of exosomes, in the present study, multilayers of gold (Au) nanoparticles were deposited by convective assembly from the gold colloidal suspension [4]. The glass substrates were further annealed at 560°C in order to modify the morphology from the multilayers of gold nanoparticles aggregates to nano-islands. In order to capture the exosomes, the affinity based synthetic polypeptide called Venceremin, Vn96 is used [6]. This molecule is specifically designed and validated to capture exosomes, having a strong affinity towards the canonical heat shock proteins (HSP), present on the surface of exosomes.The detection method is initially carried out at the substrate level, by performing the sensing protocol in a discontinuous manner. The concentrations of the chemical and biological entities at each stage of biosensing were optimized in order to facilitate the transfer to the microfluidics stage. The results indicated that this platform, is promising for the detection of exosomes. Further, in order to enhance the sensitivity of the detection and to accomplish the molecular profiling of the captured exosomes, microfluidic devices were designed, developed and tested.The results indicated that label-free technique, based on the sensitivity of the Au-LSPR band to the surrounding environment is promising for the detection of MCF-7 exosomes by the affinity approach, using the Vn96 polypeptide. This approach seems to be...

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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

Cited by 157 publications

References 49 publications

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Tumor-Related Exosomes Contribute to Tumor-Promoting Microenvironment: An Immunological Perspective

Gold Nano-Island Platform for the Microfluidic Plasmonic Detection of Exosomes

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