Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes
Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes
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DOI：
10.1016/j.ymeth.2012.01.002
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发表时间：
2012-02-01
期刊：
影响因子：
4.8
通讯作者：
Simpson, Richard J.
中科院分区：
文献类型：
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作者：
Tauro, Bow J.;Greening, David W.;Simpson, Richard J.
外来体是40-100 nm的细胞外囊泡，其从多种细胞类型释放，并执行多种细胞功能，包括细胞间通讯、抗原呈递和致癌蛋白以及mRNA和miRNA的转移。已经使用多种策略和技术从生物流体和体外细胞培养物中纯化外来体。然而，所有制备物总是含有不同比例的与外来体共纯化的其他膜囊泡，例如脱落的微囊泡和凋亡泡。使用结肠直肠癌细胞系LIM 1863作为细胞模型，在本研究中，我们对目前用于外泌体分离的方法进行了全面评估，包括超离心（UC-Exos）、OptiPrep（TM）基于密度的分离（DG-Exos）和使用抗EpCAM包被的磁珠的免疫亲和捕获（IAC-Exos）。值得注意的是，所有分离物都含有40-100 nm的囊泡，并且基于电子显微镜和蛋白质印迹，对于外来体标记物（阿利克斯、TSG 101、HSP 70）是阳性的。我们采用蛋白质组学方法来分析外泌体的蛋白质组成，并采用无标记光谱计数来评估每种方法的有效性。基于鉴定的外泌体标记物和与其生物发生、运输和释放相关的蛋白质的MS/MS谱的数量，我们发现IAC-Exos是分离外泌体的最有效方法。例如，与UG-Exos和DG-Exos相比，IAC-Exos中的阿利克斯、TSG 101、CD 9和CD 81显著更高（至少2倍）。免疫亲和捕获的应用使得能够首次在外来体中鉴定包括ESCRT-III组分VPS 32 C/CHMP 4C和SNARE突触泡蛋白2（VAMP 2）的蛋白质。此外，在IAC-Exos中鉴定了几种癌症相关蛋白，包括各种肝配蛋白（EFNB 1，EFNB 2）和Eph受体（EPHA 2 -8，EPHB 1 -4），以及参与Wnt（CTNNB 1，TNIK）和Ras（CRK，GRB 2）信号传导的组分。(C)2012 Elsevier Inc. All rights reserved.
Exosomes are 40-100 nm 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 (TM) density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100 nm 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. (C) 2012 Elsevier Inc. All rights reserved.