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Localized Proteomics of Individual Neurons Isolated from Formalin-Fixed, Paraffin-Embedded Tissue Sections Using Laser Capture Microdissection

  • Eleanor Drummond
  • Shruti Nayak
  • Beatrix Ueberheide
  • Thomas Wisniewski
Protocol
Part of the Neuromethods book series (NM, volume 127)

Abstract

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 words

Proteomics Mass spectrometry LC-MS Laser capture microdissection Alzheimer’s disease Neurons Single cell 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Eleanor Drummond
    • 1
  • Shruti Nayak
    • 2
  • Beatrix Ueberheide
    • 3
  • Thomas Wisniewski
    • 1
    • 4
  1. 1.Department of Neurology, Center for Cognitive NeurologyNew York University School of MedicineNew YorkUSA
  2. 2.Proteomics Laboratory, Division of Advanced Research and TechnologiesNew York University School of MedicineNew YorkUSA
  3. 3.Department of Biochemistry and Molecular PharmacologyNew York University School of MedicineNew YorkUSA
  4. 4.Departments of Neurology, Pathology and PsychiatryCenter for Cognitive Neurology and NYU Neuroscience Institute, New York University School of MedicineNew YorkUSA

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