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

Autor:	Griesser E; Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom; Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany., Wyatt H; Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany., Ten Have S; Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom., Stierstorfer B; Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany., Lenter M; Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany., Lamond AI; Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, United Kingdom. Electronic address: a.i.lamond@dundee.ac.uk.
Jazyk:	angličtina
Zdroj:	Molecular & cellular proteomics : MCP [Mol Cell Proteomics] 2020 May; Vol. 19 (5), pp. 839-851. Date of Electronic Publication: 2020 Mar 04.
DOI:	10.1074/mcp.RA119.001889
Abstrakt:	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. Competing Interests: * The authors declare that they have no conflicts of interest with the contents of this article. (© 2020 Griesser et al.)
Databáze:	MEDLINE
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