Proteomic Analysis of Nuclei Dissected from Fixed Rat Brain Tissue Using Expression Microdissection

Abstract

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.