Murugesan Nivetha, Macdonald Jennifer A, Lu Qiaozhan, Wu Shiaw-Lin, Hancock William S, Pachter Joel S
Department of Cell Biology, University of Connecticut Health Center, Farmington, CT, USA.
Methods Mol Biol. 2011;686:297-311. doi: 10.1007/978-1-60761-938-3_14.
The blood-brain barrier (BBB) has been well studied in terms of its pharmacological properties. However, for a better understanding of the molecular mechanisms regulating these activities, means to thoroughly investigate the BBB at the genomic and proteomic levels are essential. Global gene expression analysis platforms have, in fact, provided a venue for cataloguing the BBB transcriptome. By comparison, and largely because of technical issues, there have been few comprehensive studies of the cerebral microvasculature at the protein level. Recent advances in both microdissection techniques and proteomic analytical tools have nonetheless circumvented many of these obstacles, allowing for isolation of relatively pure cell populations from complex tissues in situ and profiling of cellular proteomes. For example, immunohistochemistry-guided laser capture microdissection (immuno-LCM) provides the unique opportunity to selectively remove brain microvascular endothelial cells from the surrounding cell populations at the BBB, while supporting downstream proteomic analysis. In this chapter, we describe the use of immuno-LCM coupled with a sensitive, high resolution, hybrid linear ion trap coupled with Fourier transform mass spectrometry (FTMS) for proteomic profiling of mouse brain microvascular endothelium, a crucial cellular component of the BBB. We provide details of the quick double-immunostaining protocol for immuno-LCM, laser capture process, sample pooling, and protein recovery followed by in-gel digestion of protein sample, mass spectrometric analysis, and protein identification. Using such an approach to obtain comprehensive protein expression profiles of the cerebral endothelium in situ will enable detailed understanding of the crucial mediators of brain microvascular signaling and BBB function in both normal and pathophysiological conditions.
血脑屏障（BBB）的药理特性已得到充分研究。然而，为了更好地理解调节这些活动的分子机制，在基因组和蛋白质组水平上彻底研究血脑屏障的方法至关重要。事实上，全球基因表达分析平台为编目血脑屏障转录组提供了一个场所。相比之下，很大程度上由于技术问题，在蛋白质水平上对脑微血管系统进行的全面研究很少。不过，显微切割技术和蛋白质组分析工具的最新进展克服了许多这些障碍，使得能够从复杂组织中原位分离出相对纯净的细胞群体并对细胞蛋白质组进行分析。例如，免疫组织化学引导的激光捕获显微切割（immuno-LCM）提供了独特的机会，可以从血脑屏障处的周围细胞群体中选择性地分离出脑微血管内皮细胞，同时支持下游的蛋白质组分析。在本章中，我们描述了使用免疫LCM结合灵敏、高分辨率的混合线性离子阱与傅里叶变换质谱（FTMS）对小鼠脑微血管内皮细胞进行蛋白质组分析的方法，脑微血管内皮细胞是血脑屏障的关键细胞组成部分。我们提供了免疫LCM快速双重免疫染色方案、激光捕获过程、样品合并和蛋白质回收的详细信息，随后对蛋白质样品进行胶内消化、质谱分析和蛋白质鉴定。使用这种方法原位获得脑内皮细胞的全面蛋白质表达谱，将有助于详细了解正常和病理生理条件下脑微血管信号传导和血脑屏障功能的关键介质。
