Cryogenic Transmission Electron Microscopy Nanostructural Study of Shed Microparticles

Issman, Liron; Brenner, Benjamin; Talmon, Yeshayahu; Aharon, Anat

doi:10.1371/journal.pone.0083680

Cited by 78 publications

(79 citation statements)

References 46 publications

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“…1F and 1G, revealed that many of the vesicles contained smaller vesicular structures within the lumen, which might represent cellular organelles or remnants thereof, or could be an artifact of MP preparation. The shapes, sizes, and the vesicles-within-vesicle feature of MP corresponded well with the nanostructures reported recently by Issman and Yuana (15,26), and confirmed successful isolation of MP from human plasma. We mathematically simulated the sedimentation behavior of MP and platelets (supplemental Fig.…”

Section: Resultssupporting

confidence: 88%

“…We believe that such an approach is in high demand to further the understanding of MP function in vascular biology and to enable for specific and sensitive screening of MP in biomarker discovery and diagnostics. MP have mostly been studied by FCM in clinical studies, although FCM is a technology known to have shortcomings in the detection of MP (12)(13)(14)(15). Nano-LC-MS/MS based proteomics is an unbiased and very sensitive analytical technology to establish an in-depth, quantitative protein parts list and to determine compositional changes, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…Their results indicated that snap freezing of PFP didn't influence MP significantly compared with direct freezing at Ϫ80°C. FCM was used to enumerate MP, which renders quantitative results ambiguous (1,(12)(13)(14)(15). Very recently, Yuana and colleagues could not find any impact on extracellular vesicle number and size after a single freeze thaw cycle of PFP (38).…”

Section: Discussionmentioning

confidence: 99%

“…Detection of MP with FCM relies on light scattering, which is dependent on refractive index, shape, and light absorption of vesicles (12). It had been shown with imaging techniques, like cryo-transmission electron microscopy (cryo-TEM) and atomic force microscopy, that FCM detects only 0.1-2% of vesicles, because of the fact that the majority of vesicles are smaller than 300 -500 nm in diameter (12)(13)(14)(15). FCM relies furthermore on cell marker specific antibodies labeled with a fluorescent dye.…”

mentioning

confidence: 99%

“…Most of former studies trying to establish best practice protocols were relying on FCM, often using Annexin-V as a marker for the enumeration of MP despite the fact that Annexin-V binds only to a subset of MP (12). Furthermore, there is no consistency in the literature of how samples were frozen and thawed, and how platelet-free plasma (PFP) was prepared before freezing (15).…”

mentioning

confidence: 99%

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Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins

Braga-Lagache

Buchs

Iacovache

et al. 2016

Molecular & Cellular Proteomics

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Cells of the vascular system release spherical vesicles, called microparticles, in the size range of 0.1-1 m induced by a variety of stress factors resulting in variable concentrations between health and disease. Furthermore, microparticles have intercellular communication/signaling properties and interfere with inflammation and coagulation pathways. Today's most used analytical technology for microparticle characterization, flow cytometry, is lacking sensitivity and specificity, which might have led to the publication of contradicting results in the past.We propose the use of nano-liquid chromatography two-stage mass spectrometry as a nonbiased tool for quantitative MP proteome analysis.For this, we developed an improved microparticle isolation protocol and quantified the microparticle protein composition of twelve healthy volunteers with a labelfree, data-dependent and independent proteomics approach on a quadrupole orbitrap instrument.Using aliquots of 250 l platelet-free plasma from one individual donor, we achieved excellent reproducibility with an interassay coefficient of variation of 2.7 ؎ 1.7% (mean ؎ 1 standard deviation) on individual peptide intensities across 27 acquisitions performed over a period of 3.5 months. We show that the microparticle proteome between twelve healthy volunteers were remarkably similar, and that it is clearly distinguishable from whole cell and platelet lysates. We propose the use of the proteome profile shown in this work as a quality criterion for microparticle purity in proteomics studies. Furthermore, one freeze thaw cycle damaged the microparticle integrity, articulated by a loss of cytoplasm proteins, encompassing a specific set of proteins involved in regulating dynamic structures of the cytoskeleton, and thrombin activation leading to MP clotting. On the other hand, plasma membrane protein composition was unaffected. Finally, we show that multiplexed data-independent acquisition can be used for relative quantification of target proteins using Skyline software. Mass spectrometry data are available via ProteomeXchange (identifier PXD003935) and panoramaweb.org (https://panoramaweb.org/labkey/

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Section: Resultssupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins

Braga-Lagache

Buchs

Iacovache

et al. 2016

Molecular & Cellular Proteomics

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Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components

et al. 2020

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Human mesenchymal stem cell exosomes have been shown to promote cutaneous wound healing. Their bioactivity is most often attributed to their protein and nucleic acid components, while the function of exosomal lipids remains comparatively unexplored. This work specifically assesses the involvement of lipids and the transmembrane enzyme CD73 in the exosomes' biological activity in stimulating the cutaneous wound healing process. Since exosome preparation processes are not harmonized yet, certain production and purification parameters are first systematically investigated, enabling the optimization of a standardized protocol delivering high exosome integrity, yield, and purity. An in situ enzymatic assay is introduced to specifically assess the vesicle functionality, and quantitative proteomics is employed to establish the cell culture conditions yielding a stable exosome protein profile. Using a combination of in vitro approaches, CD73 and constitutional lipids of HPV-16 E6/E7 transformed human bone marrow stromal cell-derived exosomes are identified as key bioactive components promoting the exosome-driven acceleration of processes required for wound repair. A pilot wound healing study in mice indeed suggests a role of lipids in the exosomes' biological activity. Strikingly, the extent of the bioactivity of these exosomal components is found to be dependent on the target cell type.

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Evaluation of exosome loading characteristics in their purification via a glycerol‐assisted hydrophobic interaction chromatography method on a polyester, capillary‐channeled polymer fiber phase

Huang

Wang

Bruce

et al. 2020

Biotechnology Progress

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Exosomes are membrane-secreted vesicles, with sizes ranging from 30 to 150 nm, which play key roles in intercellular communication. There is intense interest in developing methods to isolate and quantify exosomes toward clinical diagnostics, fundamental studies of intercellular processes, and use of exosomes as delivery vehicles for therapeutic agents. Current methods for exosomes isolation and quantification are time consuming and have operational high costs; few combine isolation and quantification into a singular operation unit. This report describes the use of hydrophobic interaction chromatography on a polyester capillary-channeled polymer fiber column, employing a step gradient for exosome elution, including use of glycerol as a solvent modifier. The entire procedure is completed in 8 min, while maintaining the structural integrity and biological activity of the isolated exosomes. Electron microscopy was used to verify the size and structural fidelity of single exosomes. Absorbance response curves for a commercial exosome sample were used for exosome quantification in the chromatographic separations. In order to determine the dynamic loading capacity for exosomes, different volumes of Dictyostelium discoideum cell culture milieu supernatant were loaded at different column lengths (5-30 cm) and loading flow rates (0.2-0.5 ml/min). A loading capacity of 5.4 × 10 12 exosomes derived from D. discoideum milieu was obtained on a 0.8 × 300 mm column; yielding recoveries of over 80%. It is believed that this isolation and purification strategy holds many advantages toward the use of exosomes across a wide breadth of medical and biotechnology applications. K E Y W O R D S capillary-channeled polymer fibers, exosomes, extracellular vesicles, hydrophobic interaction chromatography, purification 1 | INTRODUCTION Exosomes, a subset of extracellular vesicles (EVs), are membranesecreted vesicles, with sizes ranging from 30 to 150 nm, which exist in almost all cell types and body fluids and play key roles in intercellular communication and cancer progression. 1,2 Formed in the multivesicular body, exosomes encapsulate various species including proteins and genetic material, and carry along surface proteins representative of their source. Notably, tumor cells have been shown to produce and secrete exosomes in greater 3, 4 numbers than normal

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Cryogenic Transmission Electron Microscopy Nanostructural Study of Shed Microparticles

Cited by 78 publications

References 46 publications

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins

Robust Label-free, Quantitative Profiling of Circulating Plasma Microparticle (MP) Associated Proteins

Exosomes for Wound Healing: Purification Optimization and Identification of Bioactive Components

Evaluation of exosome loading characteristics in their purification via a glycerol‐assisted hydrophobic interaction chromatography method on a polyester, capillary‐channeled polymer fiber phase

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