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      Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

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          Abstract

          Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

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          Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

          Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. Copyright © 2012 Elsevier Inc. All rights reserved.
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            Large-scale proteomics and phosphoproteomics of urinary exosomes.

            Normal human urine contains large numbers of exosomes, which are 40- to 100-nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary space. Here, we used LC-MS/MS to profile the proteome of human urinary exosomes. Overall, the analysis identified 1132 proteins unambiguously, including 177 that are represented on the Online Mendelian Inheritance in Man database of disease-related genes, suggesting that exosome analysis is a potential approach to discover urinary biomarkers. We extended the proteomic analysis to phosphoproteomic profiling using neutral loss scanning, and this yielded multiple novel phosphorylation sites, including serine-811 in the thiazide-sensitive Na-Cl co-transporter, NCC. To demonstrate the potential use of exosome analysis to identify a genetic renal disease, we carried out immunoblotting of exosomes from urine samples of patients with a clinical diagnosis of Bartter syndrome type I, showing an absence of the sodium-potassium-chloride co-transporter 2, NKCC2. The proteomic data are publicly accessible at http://dir.nhlbi.nih.gov/papers/lkem/exosome/.
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              Exosome isolation for proteomic analyses and RNA profiling.

              While the existence of exosomes has been known for over three decades, they have garnered recent interest due to their potential diagnostic and therapeutic relevance. The expression and release of specific tumor-derived proteins into the peripheral circulation has served as the centerpiece of cancer screening and diagnosis. Recently, tissue-associated microRNA (miRNA) has been shown to be characteristic of tumor type and developmental origin, as well as exhibit diagnostic potential. Tumors actively release exosomes, exhibiting proteins and RNAs derived from the originating cell, into the peripheral circulation and other biologic fluids. Recently, we have demonstrated the presence of miRNAs within the RNA fraction of circulating tumor-derived exosomes. Currently, in over 75 investigations compiled in ExoCarta, over 2,300 proteins and 270 miRNAs have been linked with exosomes derived from biologic fluids. Our previous work has indicated that these circulating exosomal proteins and miRNAs can serve as surrogates for the tumor cell-associated counterparts, extending their diagnostic potential to asymptomatic individuals. In this chapter, we compare currently utilized methods for purifying exosomes for postisolation analyses. The exosomes derived from these approaches were assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, circulating exosomes isolated by ExoQuick precipitation produces exosomal RNA and protein with greater purity and quantity than chromatography, ultracentrifugation, and DynaBeads. While this precipitation approach isolates exosomes in general and does not exhibit specificity for the originating cell, the increased quantity and quality of exosomal proteins and RNA should enhance the sensitivity and accuracy of down-stream analyses, such as qRT-PCR profiling of miRNA and mass spectrometric and electrophoretic analyses of exosomal proteins.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                06 January 2015
                2015
                : 5
                : 7639
                Affiliations
                [1 ]Centre for Personalised NanoMedicine, The University of Queensland , St Lucia 4072, QLD, Australia
                [2 ]Australian Institute for Bioengineering and Nanotechnology, The University of Queensland , St Lucia 4072, QLD, Australia
                [3 ]School of Chemistry and Molecular Biosciences, The University of Queensland , St Lucia 4072, QLD, Australia
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                srep07639
                10.1038/srep07639
                4648344
                25559219
                41b81ff8-0e72-4ec6-83b9-a6a75e83fbb7
                Copyright © 2015, Macmillan Publishers Limited. All rights reserved

                This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

                History
                : 26 August 2014
                : 02 December 2014
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