Centre for Applied Pharmacokinetic Research (CAPKR), University of Manchester, Manchester, UK.
ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.
J Neurochem. 2018 Sep;146(6):670-685. doi: 10.1111/jnc.14446. Epub 2018 Aug 1.
The blood-brain barrier (BBB) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make-up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15-fold enrichment of endothelial cell marker Glut1 mRNA, whereas markers for other cell types were not enriched. Filter-aided sample preparation was shown to be superior to in-solution sample preparation (10251 peptides vs. 7533 peptides). Label-free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP-binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.
血脑屏障 (BBB) 通过严格调节与全身循环的分子交换来维持脑内稳态。它主要由微血管内皮细胞组成，周围是星形胶质细胞终足、周细胞和小胶质细胞。了解大鼠 BBB 中转运体的构成对于将药理学和毒理学观察结果转化为人类研究至关重要。在这项研究中，提出了实验工作流程，其中优化了 (a) 大鼠脑微血管的分离、(b) 内皮细胞的富集以及 (c) 蛋白质的提取和消化，随后鉴定和定量了 BBB 蛋白。内皮细胞标志物 Glut1 mRNA 的 15 倍富集表明微血管分离得到了优化，而其他细胞类型的标志物则没有得到富集。过滤辅助样品制备优于溶液内样品制备 (10251 个肽段与 7533 个肽段)。无标记蛋白质组学用于鉴定近 2000 种蛋白质，并定量分离的微血管中的 1276 种蛋白质。采用靶向和全局蛋白质组学相结合的方法来测量 6 种 ABC 转运体和 27 种溶质载体转运体的蛋白质丰度。使用专有的 Progenesis 和开放访问的 MaxQuant 软件进行数据分析显示出总体一致性；然而，Abcb9 和 Slc22a8 仅由 MaxQuant 定量，而 Abcc9 和 Abcd3 仅由 Progenesis 定量。靶向和非靶向定量之间的一致性在 Abcb1（19.7±1.4 与 17.8±2.3）和 Abcc4（2.2±0.7 与 2.1±0.4）中得到了证明。正如本研究所述，对 BBB 蛋白进行严格定量应该有助于涉及外源性物质在脑中分布的转化模型研究。
