Exosome enrichment of human serum using multiple cycles of centrifugation.
Exosome enrichment of human serum using multiple cycles of centrifugation.
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DOI：
10.1002/elps.201500131
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发表时间：
2015-09
期刊：
影响因子：
2.9
通讯作者：
Lubman DM
中科院分区：
文献类型：
--
作者：
Kim J;Tan Z;Lubman DM
在这项工作中，我们比较了使用常规速度的重复离心循环从人血清中富集外来体与使用超离心。去除细胞和细胞碎片后，分别使用110，000 ×g或40，000 ×g的速度进行超离心或离心富集过程。使用BCA测定、1-D凝胶分离、透射电子显微镜、蛋白质印迹和高分辨率LC-MS/MS分析来分析富集的外泌体。发现超离心或离心的五个循环重复对于在从人血清富集外来体中成功去除非外来体蛋白是必要的。更重要的是，发现5×离心富集在富集的外泌体蛋白量、用于检测CD-63的蛋白质印迹条带强度以及鉴定的外泌体相关蛋白和CD蛋白的数量方面提供与5×超离心富集相似或更好的性能。在对5次超离心和5次离心获得的外泌体蛋白的LC-MS/MS分析中，总共鉴定了478个蛋白，包括许多重要的CD膜蛋白。先前报道的外泌体相关蛋白（包括关键外泌体蛋白标志物）的存在证明了该方法用于分析人血清中蛋白质的实用性。
In this work, we compared the use of repeated cycles of centrifugation at conventional speeds for enrichment of exosomes from human serum compared to the use of ultracentrifugation. After removal of cells and cell debris, a speed of 110,000×g or 40,000×g was used for the ultracentrifugation or centrifugation enrichment process, respectively. The enriched exosomes were analyzed using the BCA assay, 1-D gel separation, transmission electron microscopy, Western blotting, and high resolution LC-MS/MS analysis. It was found that a five cycle repetition of ultracentrifugation or centrifugation is necessary for successful removal of non-exosomal proteins in the enrichment of exosomes from human serum. More significantly, 5×centrifugation enrichment was found to provide similar or better performance than 5×ultracentrifugation enrichment in terms of enriched exosome protein amount, Western blot band intensity for detection of CD-63 and numbers of identified exosome-related proteins and CD proteins. A total of 478 proteins were identified in the LC-MS/MS analyses of exosome proteins obtained from 5×ultracentrifugations and 5×centrifugations including many important CD membrane proteins. The presence of previously reported exosome-related proteins including key exosome protein markers demonstrates the utility of this method for analysis of proteins in human serum.