Name: biochemical purification and proteomic characterization of amyloid fibril cores from the brain
Uploaded: 2022-04-28T13:50:00
Description: Watch this Scientific Journal Video about Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain at JoVE.com

This work was supported by the NIH grant R01AG061865 to R.J.V. and J.N.S. The authors thank Vassar and Savas research group members at Northwestern University for their thoughtful discussions. We also sincerely thank Dr(s). Ansgar Seimer and Ralf Langen at the University of South California for their crucial input. We thank Dr. Farida Korabova for sample preparation and negative staining electron microscopy imaging at Northwestern University Center for Advanced Microscopy.

This protocol involves the use of human or vertebrate brain tissues. All the research was performed in compliance with the approved institutional guidelines of the Northwestern University. The current workflow is standardized using APP-knock in (AppNL-G-F/NL-G-F) mouse brain cortical and hippocampal brain region extracts11. This protocol has been optimized for brain extracts from mice at 6-9 months of age, and it can effectively purify amyloids from both male and female animals...

<ol>
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Developing a clear understanding of amyloid structure and composition is challenging for structural biologists and biochemists due to the biological complexities and experimental limitations in extracting purified fibrils from AD brain tissues16,17. Amyloid fibrils are polymorphic at the molecular level, showing a heterogeneous population of varying lengths and complexities18,19. To better understand thei...

Amyloid is an extremely stable supramolecular arrangement that is found in a diverse panel of proteins, some of which lead to pathological changes1. The accumulation of intra- or extracellular amyloid aggregates is observed in several neurodegenerative diseases2. Amyloid aggregates are heterogeneous and are enriched with a large number of proteins and lipids3. In recent years, interest in the amyloid proteome has generated substantial interest among basic and translational neuroscientists. Several methods have been developed to extract and purify amyloid aggregates from mouse and post-mortem human brain tissues. Laser-capture microdissection, immunoprecipitation, decellularization, and biochemical isolation of amyloid aggregates are widely used methods to extract and purify amyloid plaques, fibrils, and oligomers4,5,6,7. Many of these studies have focused on determining the protein composition of these tightly packed fibrillar deposits using semi-quantitative MS. However, the available results are inconsistent, and the surprisingly large number of co-purifying proteins previously reported are challenging to interpret.
The primary limitation of the existing literature describing the amyloid core proteome in AD and AD mouse model brains is that the purified material contains an unmanageable number of co-purifying proteins. The overall goal of this method is to overcome this limitation and develop a robust biochemical purification for isolating amyloid fibril cores. This strategy employs a previously described sucrose density gradient ultracentrifugation-based biochemical method for the isolation of SDS insoluble enriched amyloid fractions from post-mortem AD human and mouse brain tissues8,9. This method builds on the existing literature but goes further with ultrasonication and SDS washes to remove most of the loosely bound amyloid-associated proteins, leading to the isolation of highly purified amyloid fibrils (Figure 1). The fibrils purified by this protocol overcome several existing challenges frequently encountered in structural studies of amyloid fibrils isolated from brain extracts. Visualization of these fibrils with transmission electron microscopy (TEM) confirms the integrity and purity of the purified material (Figure 2). In this study, the isolated fibrils are solubilized and digested to peptides with trypsin, and label-free MS analysis can readily reveal the identity of the proteins forming the fibril core. Notably, some of these proteins have an inherent tendency to form supramolecular assemblies in non-membrane-bound organelles. In addition, many of the proteins identified in the analysis of amyloid-beta (A&#946;) fibrils are also associated with other neurodegenerative diseases, suggesting that these proteins may play a key role in multiple proteinopathies.
This SDS/ultrasonication method is unlikely to alter or disrupt the structure of the fibril cores. The purified material is also suitable for a wide range of top-down and bottom-up proteomic analysis approaches and additional MS-based structural analysis strategies, such as chemical crosslinking or hydrogen-deuterium exchange. The overall recovery using this method is relatively high and, thus, is suitable for detailed structural studies, which require micrograms to milligrams of the purified material. The purified material is also suitable for structural studies using cryoEM and atomic force microscopy. This protocol, in combination with the stable isotopic labeling of mammals, can facilitate solid-state nuclear magnetic resonance (NMR) studies of amyloid structure10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acclaim PepMap 100 C18 HPLC column 0.075 mm x 20&#160;mm</td>
			<td>Thermo Scientific</td>
			<td>164535</td>
			<td rowspan="35">Alternative instruments, chemicals and antibodies from other manufacturers can be used</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (1-16) 6E10 antibody</td>
			<td>Biolegend</td>
			<td>803001</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (17-24) 4G8 antibody</td>
			<td>Biolegend</td>
			<td>800701</td>
		</tr>
		<tr>
			<td>anti-amyloid beta (N terminus 82E1) antibody</td>
			<td>IBL America</td>
			<td>10323</td>
		</tr>
		<tr>
			<td>anti-amyloid fibril LOC antibody</td>
			<td>&#160;EMD Millipore</td>
			<td>AB2287</td>
		</tr>
		<tr>
			<td>BCA kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
		</tr>
		<tr>
			<td>Bioruptor Pico Plus</td>
			<td>Diagenode</td>
			<td>B01020001</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;C1016</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C0130</td>
		</tr>
		<tr>
			<td>Complete&#160; Protease Inhibitor Cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11697498001</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0521</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
		</tr>
		<tr>
			<td>HyperSep C18 Cartridges</td>
			<td>Thermo Fisher Scientific</td>
			<td>60108-302</td>
		</tr>
		<tr>
			<td>Integrated Proteomics Pipeline - IP2&#160;</td>
			<td>http://www.integratedproteomics.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
		</tr>
		<tr>
			<td>K54 Tissue Homogenizing System Motor</td>
			<td>Cole Parmer</td>
			<td>Glas-Col 099C</td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>https://www.maxquant.org/</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23235</td>
		</tr>
		<tr>
			<td>Nanoviper 75 &#956;m x 50&#160;cm</td>
			<td>Thermo Scientific</td>
			<td>164942</td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>BR-8101P-E</td>
		</tr>
		<tr>
			<td>Orbitrap Fusion TribridMass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMBCX</td>
		</tr>
		<tr>
			<td>Pierce C18 Spin Columns</td>
			<td>Thermo Fisher Scientific</td>
			<td>89870</td>
		</tr>
		<tr>
			<td>Precellys 24 tissue homogenizer</td>
			<td>Bertin Instruments</td>
			<td>P000062-PEVO0-A</td>
		</tr>
		<tr>
			<td>ProteaseMAX(TM) Surfactant Trypsin Enhancer</td>
			<td>Promega</td>
			<td>V2072</td>
		</tr>
		<tr>
			<td>RawConverter</td>
			<td>http://www.fields.scripps.edu/rawconv/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>VWR</td>
			<td>97064-646</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>74255</td>
		</tr>
		<tr>
			<td>Sorvall Legend Micro 21R Microcentrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75002446</td>
		</tr>
		<tr>
			<td>Speed Vaccum Concentrator</td>
			<td>Labconco</td>
			<td>7315021</td>
		</tr>
		<tr>
			<td>Tris-2-carboxyethylphosphine (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>C4706-2G</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Thermo Fisher Scientific</td>
			<td>15568025</td>
		</tr>
		<tr>
			<td>Trypsin Gold-Mass spec grade</td>
			<td>Promega</td>
			<td>V5280</td>
		</tr>
		<tr>
			<td>UltiMate 3000 RSLCnano System</td>
			<td>Thermo Scientific</td>
			<td>ULTIM3000RSLCNANO</td>
		</tr>
	</tbody>
</table>

biochemical purification and proteomic characterization of amyloid fibril cores from the brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

Here, a detailed method for the isolation and purification of amyloid fibrils using a modified sucrose density gradient ultracentrifugation purification method is summarized (see Figure 1). The innovation in this method is the inclusion of steps of ultrasonication-based washing using a water bath sonication system followed by SDS solubilization, which removes many loosely associated proteins from the amyloid fibrils that co-purify with the highly dense and clean fibrils. The ultrasonication ...

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Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Research

JoVE Journal

Neuroscience

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