Localized Proteomics of Individual Neurons Isolated from Formalin-Fixed, Paraffin-Embedded Tissue Sections Using Laser Capture Microdissection
Localized proteomics of specific cell populations will greatly aid understanding of the complex etiology of neurodegenerative diseases. The use of proteomics offers considerable advantages over traditional protein detection techniques and is likely to be particularly useful for studying neurodegenerative diseases, as selective vulnerability of specific neuron populations is a defining characteristic of these diseases. Pathogenesis is likely to involve complex interactions between multiple proteins. Therefore, a technique that can quantify all proteins in a cell population at the same time is preferred. Methodology that enables proteomics on cells isolated from archived formalin-fixed, paraffin-embedded (FFPE) tissue is of particular benefit for future studies because of the large FFPE tissue repositories. Here, we describe our protocol for localized proteomics of neurons microdissected from archived FFPE Alzheimer’s disease brain tissue. Neurons are visualized by staining with cresyl violet and isolated using laser capture microdissection (LCM). Collection of 1.5 mm2 total tissue area (approximately 12,000 neurons) provides enough material for quantitative mass spectrometry. The excised tissue is directly deparaffinized, reduced, alkylated, proteolytically digested, desalted, and analyzed using LC-MS.
Key wordsProteomics Mass spectrometry LC-MS Laser capture microdissection Alzheimer’s disease Neurons Single cell
This work was supported by the following NIH grants: AG08051, AG20245, and NS073502. Additional support was provided by the Seix Dow Foundation.
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