National Cancer Institute, NIH, Bethesda, Maryland 20892, United States.
Anal Chem. 2013 Aug 6;85(15):7139-45. doi: 10.1021/ac400691k. Epub 2013 Jul 19.
Expression microdissection (xMD) is a high-throughput, operator-independent technology that enables the procurement of specific cell populations from tissue specimens. In this method, histological sections are first stained for cellular markers via either chemical or immuno-guided methods, placed in close contact with an ethylene vinyl acetate (EVA) film, and exposed to a light source. The focal, transient heating of the stained cells or subcellular structures melts the EVA film selectively to the targets for procurement. In this report, we introduce a custom-designed flashcube system that permits consistent and reproducible microdissection of nuclei across an FFPE rat brain tissue section in milliseconds. In addition, we present a method to efficiently recover and combine captured proteins from multiple xMD films. 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 concurrent depletion of proteins localized to the cytoplasm, cytoskeleton, and mitochondria. These data demonstrate that the flashcube-xMD technology is applicable to the proteomic study of a broad range of targets in molecular pathology.
表达微切割（xMD）是一种高通量、操作员独立的技术，可从组织标本中获取特定的细胞群体。在这种方法中，通过化学或免疫引导方法对组织切片进行细胞标志物染色，然后将其与乙烯-醋酸乙烯酯（EVA）膜紧密接触，并暴露在光源下。染色细胞或亚细胞结构的焦点瞬态加热会选择性地将 EVA 膜融化到目标位置进行提取。在本报告中，我们介绍了一种定制设计的闪光立方体系统，该系统可在毫秒内对 FFPE 大鼠脑组织切片上的核进行一致且可重复的微切割。此外，我们还提出了一种从多个 xMD 薄膜中高效回收和组合捕获蛋白的方法。光镜和扫描电子显微镜都显示出捕获的核结构。对样品的鸟枪法蛋白质组学分析表明，核定位蛋白显著富集，平均有 25%的回收蛋白定位于核内，而整个组织对照为 15%（p < 0.001）。使用多重反应监测（MRM）的靶向质谱分析显示出更令人印象深刻的数据，组蛋白富集了 3 倍，同时细胞质、细胞骨架和线粒体定位的蛋白减少。这些数据表明，闪光立方体-xMD 技术适用于分子病理学中广泛目标的蛋白质组学研究。
