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

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.