Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
Methods. 2012 Feb;56(2):293-304. doi: 10.1016/j.ymeth.2012.01.002. Epub 2012 Jan 21.
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.
外泌体是 40-100nm 的细胞外囊泡，由多种细胞类型释放，具有多种细胞功能，包括细胞间通讯、抗原呈递以及致癌蛋白以及 mRNA 和 miRNA 的转移。已经使用多种策略和技术从生物体液和体外细胞培养物中纯化外泌体。然而，所有的制剂都不可避免地包含不同比例的其他膜性囊泡，这些囊泡与外泌体共同纯化，如脱落的微泡和凋亡泡。本研究使用结直肠癌细胞系 LIM1863 作为细胞模型，对外泌体分离的当前方法进行了全面评估，包括超速离心（UC-Exos）、OptiPrep™ 基于密度的分离（DG-Exos）和使用抗-EpCAM 包被的磁性珠的免疫亲和捕获（IAC-Exos）。值得注意的是，所有分离物均含有 40-100nm 的囊泡，并基于电子显微镜和 Western blot 检测到外泌体标志物（Alix、TSG101、HSP70）呈阳性。我们采用蛋白质组学方法对囊泡的蛋白质组成进行了分析，并采用无标记谱计数法评估了每种方法的有效性。基于外泌体标志物和与其生物发生、运输和释放相关的蛋白质的 MS/MS 谱的数量，我们发现 IAC-Exos 是分离外泌体最有效的方法。例如，与 UC-Exos 和 DG-Exos 相比，Alix、TSG101、CD9 和 CD81 在 IAC-Exos 中的含量明显更高（至少 2 倍）。免疫亲和捕获的应用使得能够首次鉴定 ESCRT-III 成分 VPS32C/CHMP4C 和 SNARE 突触融合蛋白 2（VAMP2）等蛋白质在囊泡中的存在。此外，在 IAC-Exos 中还鉴定出几种与癌症相关的蛋白质，包括各种 ephrins（EFNB1、EFNB2）和 Eph 受体（EPHA2-8、EPHB1-4）以及涉及 Wnt（CTNNB1、TNIK）和 Ras（CRK、GRB2）信号通路的蛋白质。
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