Zhang Fugeng, Zhao Xinchao, Xu Bei, Cheng Shuai, Tang Cheng, Duan Hongquan, Xiao Xuefeng, Du Wuxun, Xu Liang
Tianjin Huanhu Hospital, Tianjin, 300060, China.
Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases, Tianjin, 300060, China.
Anal Bioanal Chem. 2016 Apr;408(10):2441-8. doi: 10.1007/s00216-016-9339-9. Epub 2016 Jan 29.
Cell membrane chromatography (CMC) is a powerful tool to study membrane protein interactions and to screen active compounds extracted from natural products. Unfortunately, a large amount of cells are typically required for column preparation in order to carry out analyses in an efficient manner. Micro-CMC (mCMC) has recently been developed by using a silica capillary as a membrane carrier. However, a reduced retention of analytes is generally associated with mCMC mostly due to a low ligand (cellular membrane) capacity. To solve this common problem, in this work a silica-based porous layer open tubular (PLOT) capillary was fabricated and, to the best of our knowledge, for the first time applied to mCMC. The mCMC column was prepared by physical adsorption of rabbit red blood cell (rRBC) membranes onto the inner surface of the PLOT capillary. The effects of the PLOT capillaries fabricated by different feed compositions, on the immobilization amount of cellular membranes (represented by the fluorescence intensity of the capillary immobilized with fluorescein isothiocyanate isomer-labeled cellular membranes) and on the dynamic binding capacity (DBC) of verapamil (VP, a widely used calcium antagonist which specific interacts with L-type calcium channel proteins located on cellular membrane of rRBC) have been systematically investigated. The fluorescence intensity of the mCMC column when combined with the PLOT capillary was found to be more than five times higher than the intensity using a bare capillary. This intriguing result indicates that the PLOT capillary exhibits a higher cellular membrane capacity. The DBC of VP in the PLOT column was found to be more than nine times higher than that in the bare capillary. An rRBC/CMC column was also prepared for comparative studies. As a result, mCMC provides similar chromatographic retention factors and stability with common CMC; however, the cellular membrane consumption for mCMC was found to be more than 460 times lower than that for CMC. Graphical Abstract Comparision of mCMC chromatograms and SEM images between bare capillary and PLOT capillary.
细胞膜色谱法（CMC）是研究膜蛋白相互作用以及筛选从天然产物中提取的活性化合物的有力工具。遗憾的是，为了高效开展分析，柱制备通常需要大量细胞。微CMC（mCMC）是最近通过使用硅胶毛细管作为膜载体而开发的。然而，分析物保留率降低通常与mCMC相关，这主要是由于配体（细胞膜）容量较低。为了解决这个常见问题，在本研究中制备了一种基于硅胶的多孔层开口管（PLOT）毛细管，据我们所知，这是首次将其应用于mCMC。通过将兔红细胞（rRBC）膜物理吸附到PLOT毛细管内表面来制备mCMC柱。系统研究了由不同进料组成制备的PLOT毛细管对细胞膜固定量（以异硫氰酸荧光素异构体标记的细胞膜固定的毛细管的荧光强度表示）以及维拉帕米（VP，一种广泛使用的钙拮抗剂，它与位于rRBC细胞膜上的L型钙通道蛋白特异性相互作用）的动态结合容量（DBC）的影响。发现与PLOT毛细管结合时mCMC柱的荧光强度比使用裸毛细管时的强度高五倍以上。这一有趣的结果表明PLOT毛细管具有更高的细胞膜容量。发现PLOT柱中VP的DBC比裸毛细管中的DBC高九倍以上。还制备了rRBC/CMC柱用于比较研究。结果表明，mCMC与普通CMC具有相似的色谱保留因子和稳定性；然而，发现mCMC的细胞膜消耗量比CMC低460倍以上。图形摘要裸毛细管和PLOT毛细管之间mCMC色谱图和扫描电子显微镜图像的比较。
