Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue

Mol Cell Proteomics. 2020 May;19(5):839-851. doi: 10.1074/mcp.RA119.001889. Epub 2020 Mar 4.


Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

Keywords: FFPE; HPLC; Parkinson's disease; TMT; Tandem mass spectrometry; laser-capture microdissection; quantification; substantia nigra; tissues.

MeSH terms

  • Formaldehyde / chemistry*
  • Humans
  • Laser Capture Microdissection*
  • Neurons / metabolism
  • Paraffin Embedding*
  • Peptides / metabolism
  • Proteins / metabolism
  • Proteome / metabolism*
  • Proteomics / methods*
  • Substantia Nigra / metabolism*
  • Tissue Fixation*


  • Peptides
  • Proteins
  • Proteome
  • Formaldehyde