Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma
Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma
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DOI：
10.1002/pmic.201300282
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发表时间：
2013-11-01
期刊：
影响因子：
3.4
通讯作者：
Mathivanan, Suresh
中科院分区：
文献类型：
--
作者：
Kalra, Hina;Adda, Christopher G.;Mathivanan, Suresh
外来体是由多种细胞释放的纳米囊泡，并且在包括血液的体液中检测到。最近的研究强调了外泌体作为个性化靶向药物递送载体和作为疾病生物标志物储存库的关键应用。虽然这些研究应用已经引起了人们的极大兴趣，并且可以转化为实践，但需要评估外泌体的稳定性，并且需要优化外泌体从血浆中的分离方案。为了优化从血浆中分离外泌体的方法，我们使用正常人血浆对三种外泌体分离技术（差速离心结合超离心、上皮细胞粘附分子免疫亲和下拉和OptiPrep（TM）密度梯度分离）进行了比较评估。基于MS、Western印迹和显微镜结果，我们发现OptiPrep（TM）密度梯度方法在分离纯的外泌体群体方面是上级的，缺乏高度丰富的血浆蛋白。此外，我们评估了在各种储存条件下90天内外泌体在血浆中的稳定性。使用外泌体标记TSG101的蛋白质印迹分析显示，外泌体稳定90天。有趣的是，在细胞摄取的背景下，分离的外泌体能够与靶细胞融合，这表明它们确实具有生物活性。
Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.