Improving the depth of coverage in membrane proteomic studies through the use of lipid-based protein immobilization technology in parallel with methanol-facilitated solubilisation
Improving the depth of coverage in membrane proteomic studies through the use of lipid-based protein immobilization technology in parallel with methanol-facilitated solubilisation
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DOI：
10.1039/b9ay00267g
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发表时间：
2010-05-01
影响因子：
3.1
通讯作者：
Hariri, Robert J.
中科院分区：
文献类型：
--
作者：
Padliya, Neerav D.;Bhatia, Mohit B.;Hariri, Robert J.
基于脂质的蛋白质固定化(LPI)技术是最近开发的一个基于纳米技术框架的促进鸟枪膜蛋白质组研究的平台。蛋白脂质体是从膜蛋白制剂中产生的。这些蛋白质脂质体被固定在LPI芯片上，然后进行蛋白质降解。蛋白水解肽经SCX层析分离后进行LC/MS分析。这项研究的重点是评估特定细胞类型的膜蛋白质组的覆盖深度如何随着所使用的样品制备方法的变化而变化。对人真皮成纤维细胞(HDFS)和人骨髓间充质干细胞(BM-hMSCs)进行了两种不同的膜蛋白质组学研究：LPI技术和甲醇促进增溶。使用LC/MS技术可以从HDFS和BM-hMSCs中识别出更多的膜蛋白，使用LPI技术比使用甲醇促进的增溶技术更多。然而，当两种样品制备方法并行使用并将MS/MS数据卷积在一起时，可以对HDFS和BM-hMSCs进行膜蛋白鉴定的数量增加了近50%。因此，LPI技术是一种非常有用的高通量鸟枪膜蛋白质组学研究技术。然而，为了最大化膜蛋白质组覆盖的深度，对于给定的细胞类型，有必要同时使用多种样品制备方法。
Lipid-based protein immobilization (LPI) technology is a platform recently developed to facilitate shotgun membrane proteomic studies based on a nanotechnology framework. Proteoliposomes are generated from a membrane protein preparation. These proteoliposomes are immobilized onto an LPI chip and then subjected to proteolysis. The proteolytic peptides are then subjected to LC/MS analysis after fractionation by SCX chromatography. The focus of this study was to evaluate how the depth of coverage of the membrane proteome of a particular cell type varied as a function of the sample preparation method used. Human dermal fibroblasts (hDFs) and human bone marrow mesenchymal stem cells (BM-hMSCs) were subjected to membrane proteomic studies using two different sample preparation methods: LPI technology and methanol-facilitated solubilisation. The number of membrane proteins that could be identified from hDFs and BM-hMSCs using LC/MS was greater using LPI technology than it was using methanol-facilitated solubilisation. However, the number of membrane protein identifications that could be made for both hDFs and BM-hMSCs increased by similar to 50% when both sample preparation methods were used in parallel and the MS/MS data was convolved together. Therefore, LPI technology is a very useful technology for high-throughput shotgun membrane proteomic studies. However, in order to maximize the depth of membrane proteome coverage that can be attained for a given cell type, it is necessary to use multiple sample preparation methods in concert.