Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules
Summary
A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.
Abstract
Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson's disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.
Introduction
Every tissue consists of a heterogeneous mixture of different cell types, but the specific isolation of one cell type often is indispensable for a more precise characterization. Laser microdissection (LMD), coupling a microscope with a laser application, is a powerful tool for the specific isolation of tissue areas, single cells, or cellular substructures out of a complex composite. The application of LMD in combination with mass spectrometry (LMD-MS) has already been successfully implemented for several research questions, including isolation of DNA1, RNA2 and proteins3,4,5. In this protocol, a revised and optimized LMD-MS protocol is described for the proteomic analysis of human post-mortem brain tissue and sub-cellular components to decipher novel pathomechanisms of Parkinson's disease.
Neuromelanin is a black, nearly-insoluble pigment found in the catecholaminergic, dopamine-producing neurons of the substantia nigra pars compacta6. Together with proteins and lipids, it accumulates in organelle-like granules surrounded by a double membrane, called neuromelanin granules (NMGs)7,8,9. NMGs can be observed from the age of three years in humans increasing in quantity and density during the aging process10,11. To date, there is no definite hypothesis on neuromelanin formation, but one assumption is that neuromelanin is formed through the oxidation of dopamine12. Other hypotheses are based on enzymatic production of neuromelanin (e.g., tyrosinase)13. Neuromelanin itself was found to have a high binding affinity to lipids, toxins, metal ions, and pesticides. Based on these findings, the formation of NMGs is assumed to protect the cell from the accumulation of toxic and oxidative substances and from environmental toxins14,15. Besides this neuroprotective function, there is evidence that neuromelanin may cause neurodegenerative effects, e.g., by iron saturation and the subsequent catalysis of free radicals16,17. Furthermore, neuromelanin released during neurodegenerative processes can be decomposed by hydrogen peroxide, which could accelerate necrosis by reactive metals and other toxic compounds previously bound to neuromelanin and may contribute to neuroinflammation and cellular damage18. However, until now the exact role of NMGs in neurodegenerative processes like in the course of Parkinson's disease is not clearly understood. Still, NMGs seem to be involved in the pathogenesis of Parkinson's disease and their specific analysis is of utmost importance to unravel their role in neurodegeneration. Unfortunately, common laboratory animals (e.g., mice and rats) and cell lines lack NMGs19. Therefore, researchers especially rely on post-mortem brain tissue for their analysis. In the past, NMG isolation by density gradient centrifugation relied on the availability of high amounts of substantia nigra tissue20,21. Today, LMD presents a versatile tool to specifically isolate NMGs from human brain samples to then analyze them by LC-MS/MS.
In this protocol, an improved and automated version of a previous protocol22 is presented for the isolation of NMGs and surrounding tissue (SN), enabling a faster sample generation, higher numbers of identified and quantified proteins, and a severe reduction of required tissue amounts.
Protocol
Representative Results
Discussion
LMD is a widely applicable technique for the isolation of specific tissue areas, single cells, or subcellular structures. In the revised and automated protocol presented here, this technique is applied for the specific isolation of neuromelanin granules (NMGs) and NMG-surrounding tissue (SN). Until now, two different approaches for the isolation of NMGs out of human post-mortem brain tissue were published and widely used:
a) A discontinuous sucrose gradient consuming 1 g of substa…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by de.NBI, a project of the German Federal Ministry of Education and Research (BMBF) (grant number FKZ 031 A 534A) and P.U.R.E. (Protein Research Unit Ruhr within Europe) and Center for Protein Diagnostics (ProDi) grants, both from the Ministry of Innovation, Science and Research of North-Rhine Westphalia, Germany.
Materials
| 1,4-dithiothreitol | AppliChem | A1101 | |
| Acetonitrile | Merck | 1.00029.2500 | |
| Ammonium bicarbonate | Sigma-Aldrich | A6141 | |
| Formic acid | Sigma-Aldrich | 56302 | |
| Iodoacetamide | AppliChem | A1666,0100 | |
| Micro Tube 500 | Carl Zeiss | 415190-9221-000 | |
| Orbitrap Fusion Lumos Tribrid mass spectrometer | Thermo Fisher Scientific | IQLAAEGAAPFADBMBHQ | |
| PALM MicroBeam | Zeiss | 494800-0014-000 | |
| PEN Membrane slide | Carl Zeiss | 415190-9041-000 | |
| substantia nigra pars compacta tissue slices | Navarrabiomed Biobank (Pamplona, Spain) | ||
| Trifluoroacetic acid | Merck | 91707 | |
| Trypsin sequencing grade | Serva | 37283.01 | |
| Ultimate 3000 RSLC nano LC system | Thermo Fisher Scientific | ULTIM3000RSLCNANO | |
| Name of Software | Weblink/Company | Version | |
| FreeStyle | Thermo Fisher Scientific | 1.6 | |
| MaxQuant | https://www.maxquant.org/ | 1.6.17.0 | |
| PALMRobo | Zeiss | 4.6 pro | |
| Perseus | https://www.maxquant.org/perseus/ | 1.6.15.0 | |
| Skyline | https://skyline.ms/project/home/software/Skyline/begin.view | 20.2.0.343 | |
| XCalibur | Thermo Fisher Scientific | 4.3 |
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