Schneider Luke V, Likhte Varsha, Wright William H, Chu Frances, Cambron Emma, Baldwin-Burnett Anne, Krakow Jessica, Smejkal Gary B
Target Discovery, Inc., 4030 Fabian Way, Palo Alto, CA 94030, USA.
Int J Proteomics. 2012;2012:838630. doi: 10.1155/2012/838630. Epub 2012 Jul 12.
Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss. In this paper, we describe the development of a new method for the quantitative extraction of intact integral membrane proteins (including GPCRs) from solid metastatic ovarian tumors using pressure cycling technology in combination with a new (ProteoSolve-TD) buffer system. This new extraction buffer is compatible with immunoaffinity methods (e.g., ELISA and immunoaffinity chromatography), as well as conventional proteomic techniques (e.g., 2D gels, western blots). We demonstrate near quantitative recovery of membrane proteins EDG2, EDG4, FASLG, KDR, and LAMP-3 by western blots. We have also adapted commercial ELISAs for serum-soluble membrane protein fragments (e.g., sVEGFR2) to measure the tissue titers of their transmembrane progenitors. Finally, we demonstrate the compatibility of the new buffers with immunoaffinity enrichment/mass spectrometric characterization of tissue proteins.
整合膜蛋白在细胞信号传导、物质运输和病原体入侵中发挥着关键的生物学作用。然而，针对这类关键蛋白的定量临床检测方法仍然难以捉摸，并且通常仅限于血清可溶性细胞外片段。此外，经典的膜蛋白分析蛋白质组学方法通常涉及对可溶性片段进行蛋白水解消化，导致细胞内和细胞外片段分离以及大量信息丢失。在本文中，我们描述了一种新方法的开发，该方法使用压力循环技术结合一种新的（ProteoSolve-TD）缓冲系统，从实体转移性卵巢肿瘤中定量提取完整的整合膜蛋白（包括GPCRs）。这种新的提取缓冲液与免疫亲和方法（如ELISA和免疫亲和色谱）以及传统的蛋白质组学技术（如二维凝胶电泳、蛋白质免疫印迹）兼容。我们通过蛋白质免疫印迹证明了膜蛋白EDG2、EDG4、FASLG、KDR和LAMP-3的近乎定量回收。我们还对用于血清可溶性膜蛋白片段（如sVEGFR2）的商业ELISA进行了改进，以测量其跨膜前体的组织滴度。最后，我们证明了新缓冲液与组织蛋白免疫亲和富集/质谱表征的兼容性。
