Wu Jing, An Mingrui, Zhu Jianhui, Tan Zhijing, Chen Grace Y, Stidham Ryan W, Lubman David M
Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Department of Internal Medicine, Division of Hematology/Oncology University of Michigan, Ann Arbor, MI 48109, USA.
J Proteomics Bioinform. 2019;12(2):38-42. doi: 10.4172/0974-276X.1000494. Epub 2019 Mar 18.
Outer membrane vesicles (OMVs) are nanosized spheres secreted by bacteria that are similar to the vesicles known as exosomes, which are secreted by most mammalian cell types. In contrast to many studies focusing on optimizing methods for enriching exosomes from biological fluid, few studies have been conducted to investigate outer membrane vesicles from fecal samples. Herein, we have developed a pipeline comprised of membrane filtration and multiple cycles of ultracentrifugation (UC) to isolate OMVs from fecal samples for proteomics analysis, where multiple cycles of UC are required for removal of contaminants. By iTRAQ labeling quantitative proteomics analysis, different filter sizes (0.22 μm and 0.45 μm) were compared in terms of their performance in enriching OMVs and eliminating background fecal material. Using the 0.45 μm filter, a slightly higher protein yield was obtained but no additional contaminating proteins from bacteria were identified compared to those from the 0.22 μm filter. The 0.45 μm filter together with the multiple cycles of UC were thus used to isolate OMVs for proteomics analysis. To our knowledge, this is the first study profiling a large number of OMV proteins from fecal samples. Such capabilities may help provide valuable information in understanding the communication between the host and microbiota, which is critical in preventing cancer and disease development.
外膜囊泡（OMV）是细菌分泌的纳米级球体，与大多数哺乳动物细胞类型分泌的被称为外泌体的囊泡相似。与许多专注于优化从生物流体中富集外泌体方法的研究不同，很少有研究对外膜囊泡进行调查粪便样本。在此，我们开发了一种由膜过滤和多次超速离心（UC）组成的流程，用于从粪便样本中分离外膜囊泡以进行蛋白质组学分析，其中需要多次超速离心来去除污染物。通过iTRAQ标记定量蛋白质组学分析，比较了不同孔径（0.22μm和0.45μm）的滤膜在富集外膜囊泡和去除粪便背景物质方面的性能。使用0.45μm滤膜时，蛋白质产量略高，但与使用0.22μm滤膜相比，未发现额外的细菌污染蛋白。因此，0.45μm滤膜与多次超速离心一起用于分离外膜囊泡以进行蛋白质组学分析。据我们所知，这是第一项对粪便样本中大量外膜囊泡蛋白进行分析的研究。这些能力可能有助于为理解宿主与微生物群之间的相互作用提供有价值的信息，这对预防癌症和疾病发展至关重要。
