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 proteins^3,

Protocol

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

1. Precool the cryostat chamber.
   NOTE: Every tissue requires different cryostat temperatures, which can be found in the respective vendor protocol.
2. Clean the stainless-steel knife with 70% ethanol ....

Results

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 (

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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