Quantitative profiling of the human substantia nigra proteome from laser-capture microdissected FFPE tissue

Eva Griesser, Hannah Wyatt, Sara Ten Have, Birgit Stierstorfer, Martin Lenter, Angus I. Lamond (Lead / Corresponding author)

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)839-851
Number of pages13
JournalMolecular & Cellular Proteomics
Volume19
Issue number5
Early online date4 Mar 2020
DOIs
Publication statusPublished - 1 May 2020

Keywords

  • FFPE
  • laser-capture microdissection
  • substantia nigra
  • TMT
  • HPLC
  • Parkinson's disease
  • Quantification
  • Tandem Mass Spectrometry
  • Tissues

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