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.

The use of human brain tissue was approved by the ethics committee of the Ruhr-University Bochum, Germany (file number 4760-13), according to German regulations and guidelines. This protocol has been applied on commercially obtained substantia nigra pars compacta tissue slices. A graphical overview of the presented protocol is shown in Figure 1.
1. Tissue sectioning
<ol>
	<li>Precool the cryostat chamber. 
	NOTE: Every tissue requi...

<ol>
	<li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+profiling+of+spermatozoa+by+laser+capture+microdissection+and+low+volume-PCR.">DNA profiling of spermatozoa by laser capture microdissection and low volume-PCR.</a> PloS One. 6 (8), 22316 (2011).</li><li>Butler, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+high-quality+RNA+from+laser+capture+microdissected+human+and+rodent+pancreas.">Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.</a> Journal of Histotechnology. 39 (2), 59-65 (2016).</li><li>Eggers, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+fiber+type-specific+protein+profiles+derived+from+adult+murine+skeletal+muscle.">Advanced fiber type-specific protein profiles derived from adult murine skeletal muscle.</a> Proteomes. 9 (2), 28 (2021).</li><li>Kley, R. 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E., Tennyson, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+and+electron+microscopic+observations+of+lewy+bodies+and+melanin+granules+in+the+substantia+nigra+and+locus+caeruleus+in+parkinson&#700;s+disease.">Phase and electron microscopic observations of lewy bodies and melanin granules in the substantia nigra and locus caeruleus in parkinson&#700;s disease.</a> Journal of Neuropathology and Experimental Neurology. 24 (3), 398-414 (1965).</li><li>Zecca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+human+substantia+nigra+neuromelanin+with+lipids+and+peptides.">Interaction of human substantia nigra neuromelanin with lipids and peptides.</a> Journal of Neurochemistry. 74 (4), 1758-1765 (2000).</li><li>Fenichel, G. M., Bazelon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+neuromelanin.+II.+Melanin+in+the+brainstems+of+infants+and+children.">Studies on neuromelanin. II. Melanin in the brainstems of infants and children.</a> Neurology. 18 (8), 817-820 (1968).</li><li>Halliday, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+specific+phases+in+the+development+of+human+neuromelanin.">Evidence for specific phases in the development of human neuromelanin.</a> Journal of Neural Transmission. 113 (6), 721-728 (2006).</li><li>Zucca, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+iron,+dopamine+and+neuromelanin+pathways+in+brain+aging+and+Parkinson's+disease.">Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson's disease.</a> Progress in Neurobiology. 155, 96-119 (2017).</li><li>Carballo-Carbajal, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tyrosinase+overexpression+implicates+age-dependent+neuromelanin+production+in+Parkinson's+disease+pathogenesis.">Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson's disease pathogenesis.</a> Nature Communications. 10 (1), 973 (2019).</li><li>Zecca, L., Zucca, F. A., Wilms, H., Sulzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromelanin+of+the+substantia+nigra:+a+neuronal+black+hole+with+protective+and+toxic+characteristics.">Neuromelanin of the substantia nigra: a neuronal black hole with protective and toxic characteristics.</a> Trends in Neurosciences. 26 (11), 578-580 (2003).</li><li>Paris, I., Lozano, J., Perez-Pastene, C., Mu&#241;oz, P., Segura-Aguilar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+neurochemical+mechanisms+in+PD+pathogenesis.">Molecular and neurochemical mechanisms in PD pathogenesis.</a> Neurotoxicity Research. 16 (3), 271-279 (2009).</li><li>Zecca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromelanin+can+protect+against+iron-mediated+oxidative+damage+in+system+modeling+iron+overload+of+brain+aging+and+Parkinson's+disease.">Neuromelanin can protect against iron-mediated oxidative damage in system modeling iron overload of brain aging and Parkinson's disease.</a> Journal of Neurochemistry. 106 (4), 1866-1875 (2008).</li><li>Zar&#553;ba, M., Bober, A., Korytowski, W., Zecca, L., Sarna, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+a+synthetic+neuromelanin+on+yield+of+free+hydroxyl+radicals+generated+in+model+systems.">The effect of a synthetic neuromelanin on yield of free hydroxyl radicals generated in model systems.</a> Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 1271 (2-3), 343-348 (1995).</li><li>Karlsson, O., Lindquist, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanin+affinity+and+its+possible+role+in+neurodegeneration.">Melanin affinity and its possible role in neurodegeneration.</a> Journal of neural transmission. 120 (12), 1623-1630 (2013).</li><li>Marsden, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pigmentation+in+the+nucleus+substantiae+nigrae+of+mammals.">Pigmentation in the nucleus substantiae nigrae of mammals.</a> Journal of Anatomy. 95, 256-261 (1961).</li><li>Tribl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Subcellular+proteomics&amp;#34;+of+neuromelanin+granules+isolated+from+the+human+brain.">34;Subcellular proteomics&amp;#34; of neuromelanin granules isolated from the human brain.</a> Molecular &amp; Cellular Proteomics: MCP. 4 (7), 945-957 (2005).</li><li>Zucca, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromelanin+organelles+are+specialized+autolysosomes+that+accumulate+undegraded+proteins+and+lipids+in+aging+human+brain+and+are+likely+involved+in+Parkinson's+disease.">Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson's disease.</a> NPJ Parkinson's Disease. 4, 17 (2018).</li><li>Plum, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+characterization+of+neuromelanin+granules+isolated+from+human+substantia+nigra+by+laser-microdissection.">Proteomic characterization of neuromelanin granules isolated from human substantia nigra by laser-microdissection.</a> Scientific Reports. 6, 37139 (2016).</li><li>Tyanova, S., Temu, T., Cox, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MaxQuant+computational+platform+for+mass+spectrometry-based+shotgun+proteomics.">The MaxQuant computational platform for mass spectrometry-based shotgun proteomics.</a> Nature Protocols. 11 (12), 2301-2319 (2016).</li><li>Krey, J. 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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...

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&#39;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&#39;s disease is not clearly understood. Still, NMGs seem to be involved in the pathogenesis of Parkinson&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4-dithiothreitol</td>
			<td>AppliChem</td>
			<td>A1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Merck</td>
			<td>1.00029.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>56302</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>AppliChem</td>
			<td>A1666,0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Tube 500</td>
			<td>Carl Zeiss</td>
			<td>415190-9221-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbitrap Fusion Lumos Tribrid mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>IQLAAEGAAPFADBMBHQ</td>
			<td></td>
		</tr>
		<tr>
			<td>PALM MicroBeam</td>
			<td>Zeiss</td>
			<td>494800-0014-000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN Membrane slide</td>
			<td>Carl Zeiss</td>
			<td>415190-9041-000</td>
			<td></td>
		</tr>
		<tr>
			<td>substantia nigra pars compacta tissue slices</td>
			<td>Navarrabiomed Biobank (Pamplona, Spain)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Merck</td>
			<td>91707</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin sequencing grade</td>
			<td>Serva</td>
			<td>37283.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimate 3000 RSLC nano LC system</td>
			<td>Thermo Fisher Scientific</td>
			<td>ULTIM3000RSLCNANO</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Software</td>
			<td>Weblink/Company</td>
			<td>Version</td>
			<td></td>
		</tr>
		<tr>
			<td>FreeStyle</td>
			<td>Thermo Fisher Scientific</td>
			<td>1.6</td>
			<td></td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>https://www.maxquant.org/</td>
			<td>1.6.17.0</td>
			<td></td>
		</tr>
		<tr>
			<td>PALMRobo</td>
			<td>Zeiss</td>
			<td>4.6 pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Perseus</td>
			<td>https://www.maxquant.org/perseus/</td>
			<td>1.6.15.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Skyline</td>
			<td>https://skyline.ms/project/home/software/Skyline/begin.view</td>
			<td>20.2.0.343</td>
			<td></td>
		</tr>
		<tr>
			<td>XCalibur</td>
			<td>Thermo Fisher Scientific</td>
			<td>4.3</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser microdissection-based protocol for the lc-ms/ms analysis of the proteomic profile of neuromelanin granules

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&#39;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.

The specific isolation of NMGs and SN tissue is the most important step for the successful application of this protocol. Using the Field of View Analysis function in the vendor-provided software of the LMD, NMGs can be automatically selected in a color-dependent manner. Therefore, tissue areas containing NMGs (Figure 2A) have to be identified and a Field of View Analysis with adjusted color thresholds has to be performed, resulting in the labeling of NMGs (<strong class="xfi...

Watch this Scientific Journal Video about Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules at JoVE.com

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

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.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Our combination of laser micro dissection and mass spectrometry allows for the investigation of spatially resolved changes on the proteomic level in any kind of tissue. In our case, neuromelanin granules. Our protocol allows the proteomic analysis based on limited sample material.Which is usually a big challenge when working with postmortem brain tissue. Since single cells can be isolated to compare healthy and disease conditions on the proteomic level. This may enhance our understanding of disease mechanisms.To begin, place the tissue membrane slide in the slide holder on the robo stage with the tissue facing upwards. To acquire an overview scan of the tissue section. Use the scan function, select the area of interest from the top left and the bottom right corners.Then select scan all ROIs to perform the scan. For the automatic selection of neuromelanin granules in the current field of view. Select field of view analysis.Then select invert result, and set the threshold for the RGB channels. So that only neuromelanin granules are highlighted in red in the preview window. Now click on okay, to use the adjusted settings for the field of view.Adjust laser settings using an area of the slide covered by the membrane only. Fill the sample collection tube cap with 50 microliters of ultrapure water. And insert the cap into the collector of the robo mover.Using the software interface, position the robo mover above the robo stage two to start sample collection. Start the laser. Control energy and focus settings during the laser process and adjust the settings if necessary.Ensure proper isolation and catapulting of the isolated objects into the sample collection tube cap for at least the first 10 objects. When sampling is completed, navigate the robo mover to its starting position and remove the sample collection tube. After spinning the samples by centrification.Dry the samples for 1.5 hours in a vacuum concentrator. After performing triptic digestion as described in the text. Prepare the sample for HPLC-MS analysis by dissolving 200 to 400 nanograms of sample peptides in a defined volume of 0.1%TFA.In inert mass spectrometric glass vial inlets. If concentration determination is not applicable due to a low sample amount. Verify identical sample loading by comparing the total ion current.To begin proteomic raw data analysis. Click load for loading the raw files into the software. Click on set experiment to assign the sample names.To define group specific parameters. Add the modifications by choosing deamidation NQ, oxidation M, and carbamidomethylation N-term as variable modifications. and then add carbamidomethylation C as fixed modification.Choose trypsin as a digestion enzyme in the digestion tab. In the label free quantification tab, add the option LFQ and if more than 10 files are to be processed, choose the fast LFQ option to shorten the processing time. Ensuring that all other group specific parameters remain in factory settings.Proceed to the global parameters tab, and add the FASTA file derived from uniprot. org in the sequences tab. Modify the identifier rule accordingly.And add the taxonomy ID 9606 for Homo sapiens. For protein quantification choose unique and razor peptides. Then add the iBAQ option as a measure for protein quantification in the label free quantification tab.Ensure that all other global parameters remain in factory settings and click on start. After the analysis has been successfully performed retrieve the proteingroups. txt output.Load the proteingroups. txt file in the analysis software. Add the iBAQ values as main columns and sort all other columns according to their type.Filter out decoys and contaminants by filtering rows based on the categorical column and filter results based on valid values. Export the output in txt format for further processing. And evaluate the results regarding the research question.In the present study, 1, 898 protein groups were identified in NMGs and 1, 565 were identified in surrounding SN tissue of which 1, 384 were identified in both tissue areas. Thus 514 and 181 protein groups were exclusively identified in NMGs and SN tissue respectively. In the representative DDA measurements.Slightly higher iBAQ values in NMGs compared to SN indicated a higher abundance of the protein cytoplasmic dynien-1, heavy chain 1 in NMGs. This observation could be verified in PRM experiments for the peptide ESPEVLLTLDILK. In which a higher abundance in NMGs was detected.Based on the peak area on MS1 and MS2 level. The most important step is by far the isolation of the NMGs, as the rest of the protocol is based on this step to be properly performed. After LMD procedure, the samples can be utilized and further process for any kind of omics technique such as genomics, transcriptomics, proteomics, or lipidomics.

laser-microdissection-based-protocol-for-lc-msms-analysis-proteomic

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Neuroscience

2.1K vistas.  Ruhr-University Bochum. Nuestra combinación de microdisección láser y espectrometría de masas permite la investigación de cambios espacialmente resueltos a nivel proteómico en cualquier tipo de tejido. En nuestro caso, gránulos de neuromelanina. Nuestro protocolo permite el análisis proteómico basado en material de muestra limitado.Lo cual suele ser un gran desafío cuando se trabaja con tejido cerebral postmortem. Dado que las células individuales se pueden aislar para comparar condiciones sanas y ...