Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis

Abstract

This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (≥0.15 g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson’s disease and dementia with Lewy bodies compared to appropriate controls.

Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection

“…The peptides were stored in 20 μL of 0.1% TFA and maintained at −80 °C until the mass spectrometric measurements could be performed. The peptide concentrations were determined via amino acid analysis as previously described14. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed with 200 ng of the sample per run using an UltiMate 3000 RSLC nano LC system (Dionex, Idstein, Germany) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).…”

“…Identified proteins were also compared with proteins identified from NM granules enriched by density gradients11419. Moreover, the proteins were further characterized regarding their function and localization via the analysis of Gene Ontology data deposited at www.uniprot.org and via functional annotation clustering using DAVID2021.…”

“…However, analysis of subcellular entities permits the identification of proteins that are less abundant and enables a more focused view of NM granules-related processes. Our group has previously developed methods based on density-gradient centrifugation with reduced tissue requirements to enrich NM granules, as well as synaptosomes for proteomic analysis14. Mass spectrometric analysis of those enriched NM granules led to the identification of NM granules specific proteins for the first time.…”

“…Based on our proteomic data we hypothesized the generation of NM granules to resemble lysosome formation1. A limiting factor of those studies was obtaining sufficient numbers of individual high quality SNpc samples in required amounts (0.15 g, at least a half SNpc) to perform one experiment14. Due to this, only studies with small sample sizes could be performed which reduces statistical power of the experimental approach.…”

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

“…After sample digestion, protein concentrations were determined by amino acid analysis as described by Plum et al (Plum et al 2013). For this process, 5 ll of each digested sample was dried completely in a glass tube.…”