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>
	<li>Willbold, D., Strodel, B., Schr&#246;der, G. F., Hoyer, W., Heise, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-type+protein+aggregation+and+prion-like+properties+of+amyloids.">Amyloid-type protein aggregation and prion-like properties of amyloids.</a> Chemical Reviews. 121 (13), 8285-8307 (2021).</li><li>Rambaran, R. N., Serpell, L. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+amyloid+fibrils+by+the+use+of+an+antibody+that+recognizes+a+generic+epitope+common+to+amyloid+fibrils.">Immunoprecipitation of amyloid fibrils by the use of an antibody that recognizes a generic epitope common to amyloid fibrils.</a> PLOS ONE. 9 (8), 105433 (2014).</li><li>Kourelis, T. V., 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=A+proteomic+atlas+of+cardiac+amyloid+plaques.">A proteomic atlas of cardiac amyloid plaques.</a> JACC: CardioOncology. 2 (4), 632-643 (2020).</li><li>Mangione, P. P., 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=Increasing+the+accuracy+of+proteomic+typing+by+decellularisation+of+amyloid+tissue+biopsies.">Increasing the accuracy of proteomic typing by decellularisation of amyloid tissue biopsies.</a> Journal of Proteomics. 165, 113-118 (2017).</li><li>Rostagno, A., Neubert, T. A., Ghiso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unveiling+brain+A&#946;+heterogeneity+through+targeted+proteomic+analysis.">Unveiling brain A&#946; heterogeneity through targeted proteomic analysis.</a> Methods in Molecular Biology. 1779, 23-43 (2018).</li><li>Roher, 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=Morphology+and+toxicity+of+A&#946;-(1-42)+dimer+derived+from+neuritic+and+vascular+amyloid+deposits+of+Alzheimer's+disease.">Morphology and toxicity of A&#946;-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease.</a> Journal of Biological Chemistry. 271 (34), 20631-20635 (1996).</li><li>Lu, J. -. X., 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=Molecular+structure+of+&#946;-amyloid+fibrils+in+Alzheimer's+disease+brain+tissue.">Molecular structure of &#946;-amyloid fibrils in Alzheimer's disease brain tissue.</a> Cell. 154 (6), 1257-1268 (2013).</li><li>Tycko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-state+NMR+studies+of+amyloid+fibril+structure.">Solid-state NMR studies of amyloid fibril structure.</a> Annual Review of Physical Chemistry. 62, 279-299 (2011).</li><li>Saito, T., 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=Single+App+knock-in+mouse+models+of+Alzheimer's+disease.">Single App knock-in mouse models of Alzheimer's disease.</a> Nature Neuroscience. 17 (5), 661-663 (2014).</li><li>Meyerhoff, J., 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=Microdissection+of+mouse+brain+into+functionally+and+anatomically+different+regions.">Microdissection of mouse brain into functionally and anatomically different regions.</a> Journal of Visualized Experiments: JoVE. 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The authors have nothing to disclose.

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 ...

Amyloid plaques are required for the diagnosis of Alzheimer's disease. However, they're not sufficient. Despite this fact, they remain enigmatic due in part to challenges in characterizing their contents.Our protocol is highly efficient at isolating amyloid fibrils at high purity and is well suited for understanding the fine details of structure and composition. Knowing the protein content of amyloid plaques may help identify the targets of therapeutic intervention that may delay, interfere, or prevent their accumulation. Therefore, delaying the disease onset.This method is well suited for extracting protein deposits from degenerated brain tissues and protein aggregates in different other protein pathways. Begin by placing a freshly dissected or snap frozen brain tissue region in a two milliliter tube containing six to eight ceramic beads in freshly prepared ice cold homogenization buffer. Then grind the tissue using a bead mill homogenizer at 4, 000 rpm with two cycles of 30 seconds on and off pulse.Now add nine milliliters of ice cold homogenization buffer to the one milliliter of brain tissue homogenate and a 15 milliliter tube and seal with laboratory wax film strips. To ensure robust solubilization, keep the tube rotating overnight at four degrees Celsius. The following day, add solid sucrose to the tissue extract suspension to a final concentration of 1.2 moles.Mix well and centrifuge had 250, 000 x g for 45 minutes at four degree Celsius. After discarding the supernatant, resuspend the pellet in two milliliters of homogenization buffer containing 1.9 molar sucrose by triturating and centrifuge at 125, 000 x g for 45 minutes at four degree Celsius. After centrifugation transfer the top white solid layer to a fresh tube and solubilize one milliliter of ice cold wash buffer by petting up and down several times.Since the pellet is also enriched with amyloid fibrils, discard the aqueous middle layer and combine the pellet with the top layer for a higher yield. Centrifuge the combined fractions at 8, 000 x g for 20 minutes at four degrees Celsius. After discarding the supernatant, resuspend the pellet in one milliliter of ice cold digestion buffer and incubate at room temperature for three hours on a vortex.Centrifuge the sample again, then wash the pellet twice in one milliliter of ice cold Tris buffer and centrifuge again. After the second wash, resuspend the pellet in one milliliter of solubilization buffer by pipetting up and down and quickly centrifuge the tube at 200, 000 x g for 60 minutes at four degrees Celsius. Save the pellet, then reduce the sucrose concentration from 1.3 to one molar by adding 50 millimolar Tris buffer to the supernatant.Centrifuge the supernatant again, then dissolve both pellets and 100 microliters of Tris buffer containing 0.5%SDS. For amyloid purification, solubilize the amyloid-rich pellets using ultrasound waves in a bath sonication device for 20 cycles, then immediately centrifuge the material at 20, 000 x g for 30 minutes at four degrees Celsius. Resuspend the pellet in 500 microliters of 0.5%SDS Tris buffer and repeat the washing four more times, After the final centrifugation step, wash the pellet in 200 microliters of ultrapure water and centrifuge 20, 000 x g for 30 minutes at four degrees Celsius to remove any remaining detergent.Dissolve the final pellet containing purified amyloid fibrils in 100 microliters of ultrapure water. Dissolve the purified 100 microliters of amyloid fibrils in 400 microliters of methanol and vortex well. Then mix in 100 microliters of chloroform and vortex again.Next, add 300 microliters of ultrapure water and vortex thoroughly. After centrifugation at 12, 000 x g for two minutes at room temperature, carefully remove the top aqueous layer without disturbing the interface layer containing the protein flake and add the same volume of methanol again. Repeat the centrifugation, discard the supernatant and air dry the pellet.For digestion, dissolve the pellet in 50 microliters of guanidine hydrochloride buffer and sonicate in an ice cold water bath. Then vortex thoroughly for 45 minutes to one hour at room temperature, add 50 microliters of 0.2%surfactant solution and vortex again for another 60 minutes. Next, add one microliter of 500 millimolar TCEP and incubate for 60 minutes.Then add two microliters of 500 millimolar iodoacetamide and incubate in the dark for 20 minutes. After the incubation, quench the iodoacetamide with five microliters of TCEP solution for 15 minutes. Next, add the required volume of 50 millimolar ammonium bicarbonate solution to the tube to reduce the guanidine concentration to 1.5 moles.Also, add 1%surfactant solution to the tube. Then add trypsin and leave the tube mixing at 37 degrees Celsius overnight. The next day, add formic acid to the digested peptide solution to lower the pH to 2.0, then activate the C18 spin column by adding 200 microliters of 50%methanol solution and spinning at 1500 x g for two minutes at room temperature.Next, equilibrate the C18 column resin beds by adding 200 microliters of equilibration buffer and spinning again for two minutes. After equilibration, load the acidified peptide solution on the C18 column and centrifuge at 1500 x g for two minutes at room temperature. Reload the flow through, spin the column, and discard the second flow through.Then wash the peptides bound to the C18 resin twice with the wash buffer. Elute the peptide three times by adding 40 microliters of elution buffer and centrifuging the column. Dry the peptides in a speed vacuum concentrator by evaporating the aqueous solution.The dry pellets can be saved at 20 degree Celsius for a few weeks before the MS analysis. Congo red staining of purified amyloids documents the enrichment of the amyloid fibrils compared to the SDS soluble fraction. The SDS soluble fraction did not show the presence of amyloids.The structure of the purified fibrils confirmed the presence of nearly pure amyloid fibrils. Also, immunogold labeling confirmed the presence of amyloid beta-42 peptides. Staining using anti-amyloid beta-42 and anti-fibril antibodies showed a relative abundance of amyloid beta-42 peptides in fibrils.Purified amyloid fractions showed the presence of approximately 250 proteins, while the fraction collected before ultrasonication and SDS wash contained more than 2, 500 proteins. Fibril cores showed enrichment of proteins associated with nonmembrane bound organelle and supramolecular complexes. Amyloids are low abundance species, maybe a few picograms to few micrograms per milligrams of brain tissues.So you need to be very careful while picking up the layers, pallets or discarding the supernatant. With the development of this technique, you can now more confidently identify proteins associated with the initial amyloid seeds, characterize their structure and target them for therapeutic intervention. Therefore, preventing the onset of this deadly disease.

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Research

JoVE Journal

Neuroscience

2.9K Görüntüleme.  Northwestern University Feinberg School of Medicine. Alzheimer hastalığının teşhisi için amiloid plaklar gereklidir. Ancak, bunlar yeterli değildir. Bu gerçeğe rağmen, kısmen içeriklerini karakterize etmedeki zorluklar nedeniyle esrarengiz kalırlar.Protokolümüz, amiloid fibrilleri yüksek saflıkta izole etmede oldukça etkilidir ve yapı ve bileşimin ince ayrıntılarını anlamak için çok uygundur. Amiloid plakların protein içeriğini bilmek, birikimlerini geciktirebilecek, engelleyebilecek veya önleyebilecek terap...