Subcellular Microdissection for the Identification of Organelle Proteins

显微病理学定义了许多人类疾病，但大多数组织学观察到的亚细胞结构的化学组成在很大程度上仍未表征。目前，没有一种方法可以在不破坏细胞的情况下分离出感兴趣的亚细胞区域，从而扭曲自然发生的蛋白质相互作用。我们结合原位标记方案和随后的显微解剖与质谱直接分析亚细胞复合物和细胞器。为此，我们改进了表达微解剖（xMD），以便使用与质谱分析兼容的优化方案分离光学暗靶（化学，抗体或金属基染色），从而允许对亚细胞结构进行无监督的热塑性捕获。与激光捕获显微解剖相反，xMD中的薄膜不含染料，因此热塑性熔化和粘附所需的热量来自染色的细胞器。xMD与显微方法兼容，避免组织均质化。将xMD技术与随后的液相色谱耦合纳米喷雾质谱（LC-MS/MS）相结合，为鉴定亚细胞成分的组成和表征其在细胞中的生理功能提供了一种新颖而独特的分析方法。我们介绍了一种定制设计的闪光立方体系统，该系统允许在几毫秒内对FFPE大鼠脑组织切片进行一致且可重复的核显微解剖。光镜和扫描电子显微镜都显示了捕获的核结构。散弹枪

Microscopic pathology defines many human diseases, but the chemical composition of most histologically observed subcellular structures remains largely uncharacterized. Currently, there is no method for isolating subcellular areas of interest without disrupting the cells and thus distorting naturally occurring protein interactions. We combined in situ labeling schemes and subsequent microdissection with mass spectrometry to directly analyze subcellular complexes and organelles. To this end, we improved expression microdissection (xMD) in order to isolate optically dark targets (chemical, antibody, or metal-based stains) using optimized protocols compatible with mass spectrometric analysis to allow unsupervised thermoplastic capture of subcellular structures. In contrast to Laser Capture Microdissection, the film in xMD does not contain a dye and consequently the heat required for thermoplastic melting and adhesion is derived from the stained organelle. xMD is compatible with microscopic methodologies and avoids tissue homogenization. Combining the xMD technique with subsequent liquid chromatography coupled nano-spray mass spectrometry (LC-MS/MS) provides a novel and unique analytical method to identify the composition of subcellular components and characterize their physiological functions in cells. We introduced a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. Both light and scanning electron microscopy demonstrated captured nuclear structures. Shotgun proteomic analysis of the samples showed a significant enrichment in nuclear localized proteins, with an average 25% of recovered proteins localized to the nucleus, versus 15% for whole tissue controls (p< 0.001). Targeted mass spectrometry using multiple reaction monitoring (MRM) showed more impressive data, with a 3-fold enrichment in histones, and a concurrentdepletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. Our data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology