Analysis of mouse brain microvascular endothelium using laser capture microdissection coupled with proteomics.
Analysis of mouse brain microvascular endothelium using laser capture microdissection coupled with proteomics.
复制标题
使用激光捕获显微切割结合蛋白质组学分析小鼠脑微血管内皮。
DOI：
10.1007/978-1-60761-938-3_14
复制
发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Pachter,JoelS
中科院分区：
文献类型：
--
作者：
Murugesan,Nivetha;Macdonald,JenniferA;Lu,Qiaozhan;Wu,Shiaw-Lin;Hancock,WilliamS;Pachter,JoelS
血脑屏障（BBB）在其药理学性质方面已经得到了很好的研究。然而，为了更好地了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BBB的方法是必不可少的。事实上，全球基因表达分析平台为BBB转录组的编目提供了场所。相比之下，主要是因为技术问题，很少有全面的研究，脑微血管在蛋白质水平。然而，显微切割技术和蛋白质组学分析工具的最新进展规避了许多这些障碍，允许从复杂组织中原位分离相对纯的细胞群和分析细胞蛋白质组。例如，免疫化学引导的激光捕获显微切割（immuno-LCM）提供了从BBB的周围细胞群中选择性地去除脑微血管内皮细胞的独特机会，同时支持下游蛋白质组学分析。在这一章中，我们描述了使用免疫LCM加上一个敏感的，高分辨率的，混合线性离子阱再加上傅里叶变换质谱（FTMS）的蛋白质组分析小鼠脑微血管内皮细胞，一个重要的细胞组成部分的血脑屏障。我们提供了用于免疫LCM的快速双免疫染色方案、激光捕获过程、样品合并和蛋白质回收的详细信息，然后是蛋白质样品的凝胶内消化、质谱分析和蛋白质鉴定。使用这样的方法来获得全面的蛋白质表达谱的脑内皮细胞原位将使详细了解的关键介质的脑微血管信号和血脑屏障功能在正常和病理生理条件。
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.