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

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.

Indication of quantum mechanical electron transport in human substantia nigra tissue from conductive atomic force microscopy analysis

Neuromelanin and ferritin in dopamine neurons of the substantia nigra have a distribution and density that is similar to disordered arrays of quantum dots in photovoltaic devices, which have been experimentally shown to conduct electric energy using quantum mechanical electron transport mechanisms. Conductive atomic force microscopy tests were performed on human substantia nigra tissue at room temperature, to determine whether evidence of electron transport is present. The test results presented here provide evidence of quantum mechanical electron transport from ferritin and neuromelanin at levels that that are sufficient to cause or contribute to generation of action potentials.