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

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 (App^{NL-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.

NOTE: For a better understanding of the overall experimental procedure, see Figure 1 for a schematic of the workflow.

1. Tissue harvesting and amyloid purification

NOTE: Ideally, amyloid fibrils should be isolated from freshly dissected brain regions. However, this method also works well with snap- or flash-frozen brain tissues. Below is a brief outline of snap-freezing brain tissues for storage for use at a later time.

1. Brain tissue harvesting and storage: Dissect mouse brains and quickly harvest the amyloid-laden regions (i.e., cortex and hippocampus), followed by snap freezing in liquid nitrogen or a dry ice alcohol bath.
   NOTE: Snap freezing helps to preserve the structural attributes of the tissue's constituents. The mice are euthanized by isoflurane and cervical dislocation^12,13. It is advised to avoid using CO₂ since that can compromise brain proteome integrity.
2. Post-dissection, cut and store fresh tissues in small blocks (e.g., 5 mm x 5 mm), as this facilitates more efficient homogenization before amyloid extraction. Transfer the tissue to the sterile cryogenic vial, tighten its cap, and rapidly submerge.
3. Keep tissues frozen in ultracold conditions (i.e., -80 °C or liquid nitrogen). If extraction is to be performed a few days later, store the tissues at -20 °C and avoid freeze and thaw cycles. Thaw the frozen tissues on wet ice when ready for extraction and purification.
   ​NOTE: Direct contact of the tissues with liquid nitrogen can cause tissue and protein damage. Note that an alcohol bath can remove permanent markers from the tube labels.

2. Enrichment of SDS insoluble material

NOTE: Perform all the steps on ice and centrifuge at 4 °C, unless stated otherwise. Details of all the buffers and solutions used in this protocol are provided in Supplementary File 1. Manufacturers and catalog numbers of chemicals and instruments are provided in the Table of Materials.

1. Start with freshly dissected or snap-frozen brain tissue regions (thawed on ice) placed into a 2 mL tube containing 6-8 ceramic beads and freshly prepared ice-cold homogenization buffer (1 mL for 0.25-1 g of wet tissue mass).
2. Transfer the tubes to the bead mill homogenizer and grind the tissue contents at 4000 rpm, with two cycles of 30 s each and an interval of 30 s in between.
   NOTE: Alternatively, motorized stirrer or homogenizer systems can be used to grind and homogenize the tissues.
3. Add 9 mL of ice-cold homogenization buffer to the 1 mL of brain whole tissue homogenate in 15 mL tubes, seal with laboratory wax film strips, and rotate end-to-end overnight at 4 °C to ensure robust solubilization.
   NOTE: To remove unwanted large cellular debris and lipids, on the next morning, spin the tubes at 800 x g at 4 °C for 10 min and collect the supernatant in a fresh tube. Resuspend the remaining pellet in 2 mL of ice-cold homogenization buffer, mix well, spin again for 10 min at 4 °C, and combine the two supernatants.
4. Slowly add solid sucrose to the tissue extract suspension to a final concentration of 1.2 M, mix well, and centrifuge at 250,000 x g for 45 min at 4 °C.
5. Carefully remove and discard the supernatant using a pipette. Resuspend the pellet in 2 mL of homogenization buffer containing 1.9 M sucrose by triturating, followed by centrifugation at 125,000 x g for 45 min at 4 °C.
   NOTE: The buffer volume can be adjusted based on the amount of material recovered from the previous step. In general, 10 volumes of homogenization buffer (V/V) is ideal for this step.
6. Collect the top white solid layer using a pipette, transfer it to a fresh tube, and solubilize in 1 mL of ice-cold wash buffer by pipetting up and down several times, without introducing air bubbles.
7. Apart from the top layer, the pellet is also enriched with amyloid fibrils. For a higher yield, combine the two fractions and proceed. Carefully remove the middle aqueous layer using a pipette and discard.
8. Centrifuge the combined fractions at 8000 x g for 20 min at 4 °C. Discard the supernatant.
9. Add 1 mL of ice-cold digestion buffer to the washed pellet, resuspend, and incubate at room temperature (RT) for 3 h.
10. Centrifuge at 8000 x g for 20 min at 4 °C and remove the supernatant using a pipette.
11. Resuspend and wash (same as Step 2.8.) the digested pellet two times in 1 mL of ice-cold Tris buffer.
12. Resuspend the washed pellet in 1 mL of solubilization buffer containing 1% SDS and 1.3 M sucrose by pipetting up and down. Centrifuge quickly at 200,000 x g for 60 min at 4 °C.
    NOTE: Although the amyloid fibrils are highly resistant to detergents and denaturants, very long exposures to the detergent (1% SDS) may affect the integrity of the fibrils or remove the tightly bound proteins, which will compromise subsequent analyses. Therefore, immediately after resuspending the pellets, proceed to centrifugation.
13. Save the pellet and increase the volume of the remaining supernatant by adding 50 mM Tris buffer (in ratio 1:0.3) to reduce the sucrose concentration from 1.3 to 1 M and centrifuge one more time at 200,000 x g for 45 min at 4 °C.
14. Dissolve the two pellets in 100 µL of Tris buffer containing 0.5% SDS and pool for amyloid purification. Visible pellets are small and should appear opaque and off-white (see Figure 2A).

3. Amyloid purification

NOTE: Combine the two pellets, solubilize by pipetting until obtaining a uniform solution and proceed with the following steps of amyloid purification.

1. For complete solubilization of the amyloid-rich material present in pellets, subject the tube to mechanical shearing produced by ultrasound waves in a bath sonication device running for 30 s ON and 30 s OFF at a medium-range frequency for 20 cycles.
2. Immediately centrifuge the material at 20,000 x g for 30 min at 4 °C and resuspend the pellet in 500 µL of 0.5% SDS Tris buffer.
3. Repeat Step 3.1. and Step 3.2. four times for a total of five washes. The number of washes can be increased to improve the purity.
   NOTE: Sonication and washing steps are optimized at 0.5% SDS as higher concentrations may affect the structure, integrity, or protein composition of the fibrils. Increasing the SDS concentration at this step is not recommended.
4. After the final centrifugation step, wash the pellet in 200 µL of ultra-pure water and centrifuge at 20,000 x g for 30 min at 4 °C to remove any remaining detergent. The washed pellet containing purified amyloid fibrils appears semi-transparent in color and is difficult to see sometimes.
5. Dissolve the final pellet containing purified amyloid fibrils in 100 µL of ultrapure water. Proceed to the next step immediately for downstream processing or save the fibril suspension at -20 °C for further analysis. If frozen, thaw on ice before starting precipitation.

4. Methanol chloroform precipitation

NOTE: If the final goal is to perform protein analysis, it is recommended to desalt and remove additional non-proteinaceous impurities.

1. Add 400 µL of methanol to the purified 100 µL of amyloid fibrils in a 1.5 mL tube and vortex well.
2. Add 100 µL of chloroform to the tube and vortex well.
3. Add 300 µL of ultrapure water and vortex thoroughly. This turns the mixture cloudy due to protein precipitation.
4. Centrifuge at 12,000 x g for 2 min at RT.
5. Carefully remove the aqueous layer (i.e., top) without disturbing the interface layer containing the protein flake.
6. Add the same volume of methanol again and centrifuge at 12,000 x g for 2 min at RT.
7. Discard the supernatant using a pipette and air-dry the pellet at RT. Avoid over-drying; amyloid fraction is difficult to redissolve if over-dried.

5. Trypsin digestion

1. Dissolve the pellet in 50 µL of guanidine hydrochloride (GuHCl) buffer. Sonicate in an ice-cold water bath and heat at 95 °C for 5 min, if required.
2. Vortex thoroughly at RT for 45 min to 1 h to dissolve it completely. The pellet will not be visible after this step, and the solution looks clear in appearance.
3. Add the same volume of 0.2% surfactant solution. Solubilize at RT with vortexing for 60 min. This step enhances trypsin enzymatic activity.
4. Add 1 µL of tris-2-carboxyethylphosphine (TCEP) from the 500 mM stock solution. Incubate for 60 min.
5. Add 2 µL of 500 mM iodoacetamide (IAA) and incubate in the dark for 20 min.
6. Quench the IAA with 5 µL of TCEP solution for 15 min.
7. Add the required volume of 50 mM ammonium bicarbonate solution to the tube to reduce the guanidine concentration to 1.5 M.
8. Add 1% surfactant solution to the tube (1 µL/50 µg of protein).
9. Add trypsin (1 µg/100 µg of protein) and leave the tube mixing at 37 °C overnight.

6. Peptide cleanup

1. Next morning, acidify the digested peptide solution by lowering the pH to 2.0 by adding formic acid.
2. Activate the C18 (n-octadecyl) spin column in a 2 mL receiver tube by adding 200 µL of 50% methanol solution and spin at 1500 x g for 2 min at RT. Repeat the activation step.
3. Equilibrate the C18 column resin beds by adding 200 µL of equilibration buffer and spinning for 2 min with the same conditions. Repeat this step.
4. Load the acidified peptide solution on the C18 column and centrifuge at 1500 x g for 2 min at RT. Collect the flow-through and reload it one more time. Discard the second flow-through.
5. Wash the peptides bound to the C18 resin with the wash buffer 2x, as done in Step 6.3. Use the same equilibration buffer for washing the column.
6. Elute the peptides by adding 40 µL of elution buffer followed by centrifugation at 1500 x g, for 2 min at RT. Repeat the elution step three times to increase the yield of the recovered peptides.
7. Dry the peptides in a speed vacuum concentrator by evaporating the aqueous solution. Dry pellets can be saved at -20 °C for a few weeks before the MS analysis.

7. Setting up mass spectrometer for peptide analysis

NOTE: For MS parameters, see Supplementary File 1 (adapted from a previous publication from the lab)¹⁴.

1. Before loading the peptide samples for the MS analysis, dissolve the dry peptide pellets in 20 µL of sample loading buffer and perform micro BCA to quantify the concentration of recovered peptides.
2. Transfer the dissolved peptides in a glass vial and load 3 µg (quantified from micro BCA) of peptides into the UPLC system autosampler.
3. Load the samples onto a vented trap column (C18 HPLC column, 0.075 mm x 20 mm) with a flow rate of 250 nL/min.
4. Arrange the trap column in line with an analytical column (0.075 µm x 500 mm) and assemble an emitter tip to the electrospray ionization (ESI) source subjected to a spray voltage of 2000 V.
   NOTE: In this study, ESI is performed, in which the mobile phase is subjected to high-voltage ionization into the gas phase. The spray created by this is directed to the vacuum chamber of the MS through a heated capillary. While under vacuum, the de-solvation of droplets and ejection of ions occurs in the presence of heat and voltage. Thereafter, the ions are accelerated toward the mass analyzer in the presence of a high voltage environment.
5. Analyze the samples with 2 h acquisition runs. This study is performed using data-dependent acquisition with the most intense top 20 precursor ion selection paradigm.
   NOTE: In data-dependent acquisition, a limited number of precursor peptides are detected in the MS1 scan and subjected to fragmentation for MS2 analysis. However, the dynamic exclusion is problematic here since highly abundant Aβ peptides will be omitted for fragmentation and result in an underestimate of their quantity.
6. For absolute quantification of Aβ peptides, run the same samples one more time using the targeted MS/MS method. For this study, an exhaustive list of all m/z ratios for various possible tryptic Aβ peptides for the APP knock-in mouse models is prepared (see Supplementary Table 1). Other groups should prepare a similar list in accordance with the available mouse model and the possible peptides generated from the protein of interest (e.g., Aβ for AD).
   NOTE: To address the exclusion of highly abundant peptides, use a targeted approach by providing a list of selected m/z values corresponding to Aβ tryptic peptides. This approach addresses the problem of data-dependent exclusion of Aβ peptides and can quantify the absolute quantities of different monomers (Aβ38, 40, and 42 peptides) with high resolution.
7. The mass spectrometer generates MS spectra of the peptide samples and saves raw data files on the target directory. Use this file to perform spectral matching using well-established statistical and bioinformatics tools. Multiple online and offline database search and analysis tools are available.

8. MS data analysis

1. Extract the MS1 and MS2 files using the offline Rawconverter tool (http://www.fields.scripps.edu/rawconv/).
2. Perform identification, quantification, and detailed analyses of the data on a web-based MS data search engine. In this study, Integrated Proteomics Pipeline – IP2 (Bruker: http://www.integratedproteomics.com/) was used.
   NOTE: Several other online and offline MS data analysis tools are available and can also be used, such as MaxQuant (https://www.maxquant.org/).
3. After uploading the MS1 and MS2 files, select an updated mouse proteome database. Here, an updated mouse database containing additional App knock-in specific mutations is selected for the identification of peptides using ProLuCId and SEQUEST algorithms¹¹.
4. In the IP2 analysis system, select the default parameters and modify them according to the experimental requirements. In this study, a peptide mass tolerance of 50 ppm for precursor and 600 ppm for fragments is used (refer to Supplementary File 1 for other parameters used in IP2).
   NOTE: Researchers can modify the search parameters in accordance with the experimental objectives. For example, for identifying post-translational modifications, add differential modification values for various PTMs (e.g., ubiquitination, SUMOylation, phosphorylation).