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Neuron Enriched Nuclear Proteome Isolated from Human Brain

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‡ ⊥ Department of Human Genetics, Department of Biochemistry, §Department of Pathology and Laboratory Medicine, Department of Pharmacology, Department of NeurologyEmory University School of Medicine, Atlanta, Georgia 30322, United States
Cite this: J. Proteome Res. 2013, 12, 7, 3193–3206
Publication Date (Web):May 24, 2013
https://doi.org/10.1021/pr400246t
Copyright © 2013 American Chemical Society

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    The brain consists of diverse cell types including neurons, astrocytes, oligodendrocytes, and microglia. The isolation of nuclei from these distinct cell populations provides an opportunity to identify cell-type-specific nuclear proteins, histone modifications, and regulation networks that are altered with normal brain aging or neurodegenerative disease. In this study, we used a method by which intact neuronal and non-neuronal nuclei were purified from human post-mortem brain employing a modification of fluorescence activated cell sorting (FACS) termed fluorescence activated nuclei sorting (FANS). An antibody against NeuN, a neuron specific splicing factor, was used to isolate neuronal nuclei. Utilizing mass spectrometry (MS) based label-free quantitative proteomics, we identified 1755 proteins from sorted NeuN-positive and negative nuclear extracts. Approximately 20% of these proteins were significantly enriched or depleted in neuronal versus non-neuronal populations. Immunoblots of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts confirmed that distinct members of the major nucleocytoplasmic structural linkage complex (LINC), nesprin-1 and nesprin-3, were differentially enriched in neurons and astrocytes, respectively. These comparative proteomic data sets also reveal a number of transcription and splicing factors that are selectively enriched in a cell-type-specific manner in human brain.

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