The authors would like to thank Agilent for their technical assistance. Kate Markham and Rafael Palomino are credited for their initial help in the synthesis and purification of the A&#946; peptide and Dr Hsiau-Wei Lee is thanked for his help in preparing Figure 1 of the manuscript.

1. Preparative HPLC Purification of the A&#946;40 or A&#946;42 Peptide
<ol>
	<li>Prepare the following buffers for the HPLC purification.
	<ol>
		<li>Prepare buffer A (20 mM NH4OH) by adding 1.3 mL of NH4OH (28% solution) to 1,000 mL of ultrapure water.</li>
		<li>Prepare buffer B (80% acetonitrile with 20 mM NH4OH) by adding 1.3 mL of NH4OH (28% solution) to a solution of 800 mL of HPLC-grade acetonitrile and 200 mL of ultrapure water.</li>
		<li>Prepare sample dissolution buffer (0.1% NH4OH) by adding 100 &#181;L of NH4OH (28% solution) to 100 mL of ultrapure water.</li>
	</ol>
	</li>
	<li>Setup the HPLC instrument as shown in Figure 1.
	<ol>
		<li>Fit the solvent bottles that contain buffer A and buffer B to the inlets of the HPLC pump using polymer tubing. Attach the polymer tubing to the HPLC pump with a one-piece fitting. Ensure that the polymer tubing of each buffer is fitted to the correct inlet valve of the instrument. Fit the HPLC pump with a degasser.</li>
		<li>Couple the HPLC pump with the inlet of the 300 &#197; 8 &#181;m 25 mm &#1093; 300 mm preparative column (Figure 1B, far left column, see Materials List) using polymer tubing.
		<ol>
			<li>Attach the polymer tubing to the preparative column with a one-piece finger tight fitting. Ensure that the polymer column is orientated in the correct manner. 
			NOTE: The stationary phase of the preparative column is comprised of poly(styrene-divinylbenzene) particles. The correct orientation of the polymer column is marked on the outer casing with a single directional arrow.</li>
		</ol>
		</li>
		<li>Connect the outlet of the column to the dual wavelength detector using polymer tubing and set the wavelength detector to 214 nm and 280 nm.
		<ol>
			<li>Alter the wavelength by changing the detection wavelength parameters in the setup instrument method option of the built-in HPLC software.</li>
		</ol>
		</li>
		<li>Attach the polymer tubing to the inlet of the wavelength detector with a one-piece finger tight fitting. Attach polymer tubing to the output valve of the wavelength detector. Attach the polymer tubing to the outlet valve of the HPLC detector with a one-piece finger tight fitting. This will be the sample collection tubing. 
		NOTE: The lack of a strong chromophore on the A&#946; peptide dictates that 214 nm be used as the primary ultraviolet (UV) wavelength for peak collection.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55482/55482fig1.jpg" /> 
Figure 1: Experimental setup of the HPLC instrument used for purification of the amyloid beta peptides. (A) The quaternary HPLC pump fitted with a degasser and variable wavelength detector set to 214 nm and 280 nm; (B) HPLC columns used for purification of the amyloid beta peptides, from left to right, 25 x 300 mm2 preparative column, 7.5 x 300 mm2 semi preparative column and 4.6 x 250 mm2 analytical column; (C) Manual injector with 20 &#181;L stainless steel injection loop used for analytical HPLC; (D) Manual injector with 10 mL stainless steel injection loop used for preparative and semi preparative purification. <a href="https://cloudfront-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Program the HPLC software to run the purification method as shown in Table 1. Enter the purification method by changing the solvent timetable parameter (in the setup instrument method option built into the HPLC software). Turn on the HPLC pump by clicking the &#34;on&#34; button on the HPLC software. 
	NOTE: The pump will begin supplying starting ratio of buffer A and buffer B through the preparative column and the HPLC instrument.
	<ol>
		<li>Leave the system for 30 min to fully equilibrate.</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Time / min</td>
			<td>% of Buffer Aa</td>
			<td>% of Buffer Bb</td>
			<td>Flow Rated / mL min-1</td>
		</tr>
		<tr>
			<td>0</td>
			<td>80</td>
			<td>20</td>
			<td>6</td>
		</tr>
		<tr>
			<td>45</td>
			<td>75.5</td>
			<td>24.5</td>
			<td>6</td>
		</tr>
		<tr>
			<td>45.01</td>
			<td>80</td>
			<td>20</td>
			<td>6</td>
		</tr>
		<tr>
			<td>52.01</td>
			<td>80</td>
			<td>20</td>
			<td>6</td>
		</tr>
		<tr>
			<td>52.02</td>
			<td>73</td>
			<td>27</td>
			<td>6</td>
		</tr>
		<tr>
			<td>85</td>
			<td>73</td>
			<td>27</td>
			<td>6</td>
		</tr>
		<tr>
			<td>92</td>
			<td>5</td>
			<td>95c</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Table 1: Timetable for the purification of the A&#946;42 and A&#946;40 peptides using the 25 &#215; 300 mm polymer column. aBuffer A - H2O with 20 mM NH4OH; b80% MeCN / 20% H2O with 20 mM NH4OH; cRan for 15 min to wash the column prior to the next injection of sample; dIn order to run a flow rate of 6 mL/min on the HPLC instrumentation, the pressure limit needs to be reduced to 200 bar.
<ol start="4">
	<li>Purification of the A&#946; peptide sample 
	Note: The crude peptide was obtained through automated solid-phase peptide synthesis.10
	<ol>
		<li>Dissolve 3 mg of crude A&#946; peptide in 4 mL of the sample dissolution buffer. Sonicate the sample for 30-60 s at room temperature and at a frequency of 40 kHz to aid dissolution.</li>
		<li>Inject the entire sample onto the HPLC column using a 5 mL plastic syringe fitted with a 16-gauge stainless steel needle. Run the purification method as outlined in sub-step 1.3. 
		NOTE: The system enables sample injection to be done through the use of a manual injector fitted with a 10 mL stainless steel injection loop (Figure 1D). The desired A&#946; peptide will elute between 72 and 74 min as a sharp resolved peak (Figure 2C).</li>
		<li>Collect the sample into a 50 mL conical centrifuge tube. Confirm the identity of the A&#946; peak through direct injection mass spectrometry of the collected eluent.11 Store the eluent for up to 12 h at -20 &#176;C. 
		NOTE: Storage of the solution for periods longer than 12 h is not advised due to the potential for oxidation of the peptide.</li>
		<li>Isolate the purified peptide by flash freezing the collected aliquot/aliquots of the A&#946; peptide in liquid nitrogen and lyophilize. Perform lyophilization by freeze-drying the sample at a temperature of -60 &#176;C and a pressure of 20 mTorr for a period of 24 h.</li>
		<li>Run the analytical HPLC protocol as outlined below to determine the purity of the A&#946; peptide. Store peptides in their lyophilized form at -20 &#176;C for a period of up to 6 months.</li>
	</ol>
	</li>
</ol>
2. Analytical HPLC Analysis of the Purified A&#946; Protein
<ol>
	<li>Prepare the HPLC buffers as outlined in sub-section 1.1. of the above protocol.</li>
	<li>Setup the analytical HPLC as per step 1.2.1 and as depicted in Figure 1 with the 4.6 &#215; 250 mm analytical column (Figure 1B, far right column) and the manual injector with 20 &#181;L stainless steel injection loop (Figure 1C) fitted to the instrument.</li>
	<li>Program the HPLC software to run the analytical method as shown in Table 2 following instructions similar to those in step 1.3.</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Time / min</td>
			<td>% of Buffer Aa</td>
			<td>% of Buffer Bb</td>
			<td>Flow Rate / mL min-1</td>
		</tr>
		<tr>
			<td>0</td>
			<td>95</td>
			<td>5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>30</td>
			<td>50</td>
			<td>50</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 2: Timetable for the HPLC purity analysis of the A&#946; peptide. aBuffer A - H2O with 20 mM NH4OH; b80% MeCN / 20% H2O with 20 mM NH4OH.
<ol start="4">
	<li>Purity analysis of the A&#946; peptide
	<ol>
		<li>Prepare a 1 mg/mL solution of the purified peptide through dissolution of the peptide in the sample buffer solution. 
		NOTE: The buffer recipe can be found in sub-section 1.1. Protein concentration is determined by measuring the protein absorption at 280 nm (A280nm).12 The molar extinction coefficient (&#949;) used to determine concentration is &#949; = 1,490 dm3 mol-1 cm-1.13</li>
		<li>Inject 20 &#181;L of the 1 mg/mL (222 &#181;M) solution onto the HPLC column and run the analytical method that was setup in step 2.3. 
		NOTE: The remaining solution not used for analysis can be flash frozen in liquid nitrogen and lyophilized to recover the A&#946; peptide. Lyophilization details can be found in sub-section 1.4.4. The A&#946; peptide will elute from the analytical column between 16 and 18 min (Figure 2D). Use the built-in integration analysis software that accompanies the HPLC instrument to determine the purity of the A&#946; peptide. Purity is determined by integrating each of the individual peaks on the spectrum and calculating peptide-peak percentage area. Typically, a purification of &#62;95% should be ascertained.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55482/55482fig2.jpg" /> 
Figure 2: Representative HPLC traces of A&#946;42. (A) Traditional C4 silica purification, conditions: Buffer A: H2O with 0.1% trifluoroacetic acid (TFA), buffer B: MeCN (acetonitrile) with 0.1% TFA, gradient: 20 to 27% buffer B over 40 min followed by isocratic 27% buffer B; (B) Preparative purification using the 25 x 300 mm2 polymer column, conditions: Buffer A: H2O with 20 mM NH4OH, buffer B: 80% MeCN / 20% H2O with 20 mM NH4OH, gradient: 20 to 27% buffer B over 70 min followed by isocratic 27% buffer B; (C) Optimized preparative purification using the 25 x 300 mm2 polymer column, conditions are described in Table 1 located in sub-section 1.3 of the protocol description text; (D) Analytical HPLC using the 4.6 x 250 mm2 polymer column, conditions: Buffer A: H2O with 20 mM NH4OH, buffer B: 80% MeCN / 20% H2O with 20 mM NH4OH, gradient-5 to 50% buffer B over 30 min. For parts A, B and C the peak corresponding to A&#946;42 is marked by an asterisk. Collection of the marked A&#946;42 peak in part C reveals a purity of &#62;95% as shown in part D. Mass spectrometry was used to determine the identity of the A&#946;42 peak. <a href="https://cloudfront-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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	<li>Querfurth, H. W., LaFerla, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+Disease.">Alzheimer's Disease.</a> N. Engl. J. Med. 362 (4), 329-344 (2010).</li><li>McGowan, 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=A&#946;42+Is+Essential+for+Parenchymal+and+Vascular+Amyloid+Deposition+in+Mice.">A&#946;42 Is Essential for Parenchymal and Vascular Amyloid Deposition in Mice.</a> Neuron. 47 (2), 191-199 (2005).</li><li>Gong, Y., 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=Alzheimer's+disease-affected+brain:+Presence+of+oligomeric+A&#946;+ligands+(ADDLs)+suggests+a+molecular+basis+for+reversible+memory+loss.">Alzheimer's disease-affected brain: Presence of oligomeric A&#946; ligands (ADDLs) suggests a molecular basis for reversible memory loss.</a> Proc. Natl. Acad. Sci. USA. 100 (18), 10417-10422 (2003).</li><li>Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Oligomers+of+the+Amyloid+&#946;-Protein+Impair+Synaptic+Plasticity+and+Behavior.">Soluble Oligomers of the Amyloid &#946;-Protein Impair Synaptic Plasticity and Behavior.</a> Behav Brain Res. 192 (1), 106-113 (2008).</li><li>Zagorski, M. G., Yang, J., Shao, H., Ma, K., Zeng, H., Hong, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+and+Chemical+Factors+Affecting+Amyloid+&#946;+Peptide+Amyloidogenicity.">Methodological and Chemical Factors Affecting Amyloid &#946; Peptide Amyloidogenicity.</a> Methods Enzymol. 309, 189-204 (1999).</li><li>Kim, W., Hecht, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+Enhance+the+Aggregation+Propensity+of+the+Alzheimer's+A&#946;+Peptide.">Mutations Enhance the Aggregation Propensity of the Alzheimer's A&#946; Peptide.</a> J Mol Bio. 377 (2), 565-574 (2008).</li><li>Fezoui, Y., 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=An+improved+method+of+preparing+the+amyloid+beta-protein+for+fibrillogenesis+and+neurotoxicity+experiments.">An improved method of preparing the amyloid beta-protein for fibrillogenesis and neurotoxicity experiments.</a> Amyloid. 7 (3), 166-178 (2000).</li><li>Zhelev, N. Z., Barratt, M. J., Mahadevan, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+reversed-phase+high-performance+liquid+chromatography+on+polystyrene-divinylbenzene+columns+for+the+rapid+separation+and+purification+of+acid-soluble+nuclear+proteins.">Use of reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene columns for the rapid separation and purification of acid-soluble nuclear proteins.</a> J Chromatogr A. 763 (1-2), 65-70 (1997).</li><li>Thess, A., 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=Sequence-engineered+mRNA+Without+Chemical+Nucleoside+Modifications+Enables+an+Effective+Protein+Therapy+in+Large+Animals.">Sequence-engineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals.</a> Mol Ther. 23 (9), 1456-1464 (2015).</li><li>Warner, C. J. A., Dutta, S., Foley, A. R., Raskatov, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+D-glutamate+at+a+critical+residue+of+A&#946;42+stabilizes+a+pre-fibrillary+aggregate+with+enhanced+toxicity.">Introduction of D-glutamate at a critical residue of A&#946;42 stabilizes a pre-fibrillary aggregate with enhanced toxicity.</a> Chem Eur J. 22 (34), 11967-11970 (2016).</li><li>Thompson, J. A., Lim, T. K., Barrow, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-line+High-performance+Liquid+Chromatography/Mass+Spectrometric+Investigation+of+Amyloid-&#946;+Peptide+Variants+Found+in+Alzheimer's+Disease.">On-line High-performance Liquid Chromatography/Mass Spectrometric Investigation of Amyloid-&#946; Peptide Variants Found in Alzheimer's Disease.</a> Rapid Commun. Mass Spectrom. 13 (23), 2348-2351 (1999).</li><li>Layne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+turbidimetric+methods+for+measuring+proteins.">Spectrophotometric and turbidimetric methods for measuring proteins.</a> Met. Enzymology. 3, 447-455 (1957).</li><li>Ioannou, J. C., Donald, A. M., Tromp, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+secondary+structure+changes+occurring+in+high+density+systems+of+BLG+dissolved+in+aqueous+pH+3+buffer.">Characterizing the secondary structure changes occurring in high density systems of BLG dissolved in aqueous pH 3 buffer.</a> Food Hydro. 46, 216-225 (2015).</li><li>Rahimi, F., Maiti, P., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-Induced+Cross-Linking+of+Unmodified+Proteins+(PICUP)+Applied+to+Amyloidogenic+Peptides.">Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides.</a> J. Vis. Exp. (23), e1071 (2009).</li><li>Bitan, G., Kirkitadze, M. D., Lomakin, A., Vollers, S. S., Benedek, G. B., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+(A&#946;)+assembly:+A&#946;40+and+A&#946;42+oligomerize+through+distinct+pathways.">Amyloid &#946;-protein (A&#946;) assembly: A&#946;40 and A&#946;42 oligomerize through distinct pathways.</a> Proc. Natl. Acad. Sci. USA. 100 (1), 330-335 (2003).</li></ol>

The authors have nothing to disclose.

The HPLC purification of the A&#946; peptide is highly dependent upon the choice of both the stationary phase employed in the purification and the mobile phase chosen to elute the peptide. The low isoelectric point of the peptide and high propensity for aggregation render traditional chromatographic conditions for the separation of hydrophobic proteins (C4 or C8 stationary phase coupled with an acidic mobile eluent) challenging, with the A&#946; peptide eluting as a prolonged broad, non-resolved peak (Figure 2A).
To circumvent this issue, the PLRP-S stationary phase, chemically stable at high pH was found to be effective for the purification of the A&#946; peptide (Figure 2B and 2C). The use of a high pH mobile phase minimized the degree of aggregation, and when optimized, gave rise to a sharp, well-resolved peak. In the optimized protocol, the percentage of buffer B was rapidly switched from 20% to 27% at the 52-minute time point and as a result, gave rise to a well-defined A&#946; peak (Figure 2C). This optimized protocol relies on all of the hydrophobic impurities being eluted from the column during the initial 20 to 24.5% increase in buffer B concentration. If impurities are found which are of a similar hydrophobicity as the A&#946; peptide, then further optimization of the HPLC protocol may be warranted. The optimized method was highly reproducible amongst individual batches of synthesized A&#946; and was capable of purifying both 40 and 42 variants of the peptide without any changes to the optimized procedure (Figure 3). For both peptides, the purity as determined by analytical HPLC was found to be greater than 95%.
Given the reports of trace impurities altering the aggregation propensity of the A&#946; peptide, it is advisable that an orthogonal biophysical characterization be employed to confirm that the purified peptide is able to undergo oligomerization. Using previously reported protocols we chose to perform PICUP.14,15 PICUP analysis of the purified proteins demonstrates the characteristic monomer through hexamer population distribution for the A&#946;40 peptide and the monomer through heptamer distribution associated with the A&#946;42 peptide.10 These results confirm that purification of the A&#946; peptides using the PLRP-S stationary phase and a high pH mobile eluent results in A&#946; proteins that are capable of aggregation.
The purification procedure reported is designed to be able to purify up to 3 mg of the A&#946; peptide in a single chromatographic run. For this procedure, it is critical to set the flow rate of the HPLC instrument at 6 mL/min. If lower flow rates of the HPLC pump are used, the HPLC run time should be extended to accommodate this. Conversely, the use of higher flow rates may warrant a reduction in the HPLC run time. However, reduction of the run time can result in the co-elution of the A&#946; peak with other peaks and therefore lower the purity of the A&#946; peptide collected. Furthermore, the HPLC instrumentation and size of the chromatographic column used for purification can greatly affect the amount of material that can be purified in a single run. If no peak is produced at the expected eluent time of the A&#946; peptide, and a large peak is produced at the eluent time corresponding to the solvent front, then too much material was initially loaded onto the column. Reducing the amount of material injected onto the HPLC column for each run will circumvent this problem. Once the A&#946; peptide has been purified, it is highly recommended that the solution be lyophilized as quickly as possible. Prolonged storage of the peptide in solution can lead to oxidation of A&#946; and therefore formation of impurities. The purity of the A&#946; peptide should always be determined by analytical HPLC. Trace amounts of impurities can alter the aggregation propensity of the peptide dramatically. If the analytical HPLC trace shows the presence of impurities, then the sample should be re-subjected to the HPLC purification protocol and its purity re-determined by analytical HPLC.
It is hoped that this tailored procedure for the purification of the A&#946;42 and A&#946;40 peptides will be of benefit for the scientific community and allow users to obtain high purity A&#946; capable of oligomer formation. It would be expected that this procedure could be adapted to other amyloidogenic peptides that are difficult to isolate and purify.

Alzheimer&#39;s disease is a neurodegenerative disorder that effects over 35 million people worldwide.1 Implicated strongly in the onset and development of the disease, is the highly aggregation prone, hydrophobic peptide Amyloid beta (A&#946;).2 A&#946; ranges from 36 to 43 amino acids in length, however, it is thought that the 42-amino acid variant, amyloid beta 42 (A&#946;42), is the most toxic form of the protein.3 This is due in most part to the ability of A&#946;42 to readily form diffusible, oligomeric species that are believed to be particularly neurotoxic entities.4 In order to further our understanding of the A&#946; peptide, it is essential to routinely obtain high purity material. The presence of trace impurities has been shown to dramatically alter the aggregation propensity properties of the peptide.5
Traditionally, the high performance liquid chromatography (HPLC) separation of hydrophobic peptides such as A&#946; has been done through the use of a combination of C4 or C8 silica-based stationary phases and an acidic mobile phase.6 However, such conditions can present a challenge to the purification of the peptide. The low isoelectric point of the A&#946; peptide (pI approximately 5.5)7 means that under acidic conditions, peptide aggregation is increased and as a result broad, non-resolved HPLC peaks that are often difficult to isolate are produced (Figure 2A). Furthermore, such broad peaks often contain impurities which may impact the aggregation profile of the peptide, and commonly require subsequent rounds of purification which can dramatically impact the amount of peptide produced.
The poly(styrene/divinylbenzene) stationary phase, PLRP-S, represents an alternative means of purifying hydrophobic peptides. The stationary phase has been employed in the purification of a number of different proteins and messenger ribonucleic acids (mRNA).8,9 The PLRP-S stationary phase requires no additional alkyl ligand for reverse phase separation, and more importantly is chemically stable at high pH which leads to deaggregation of the peptide.7 Herein, we report a tailored HPLC purification protocol that yields high purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides.

<table><tbody><tr><td>Agilent 1260 Infinity II quarternary pump</td><td>Agilent</td><td>G7111B</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump</td></tr><tr><td>Agilent 1260 Infinity II Dual variable wavelength detector</td><td>Agilent</td><td>G7114A</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop</td><td>Agilent</td><td>0101-1232</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Agilent 1260 Infinity II Manual Injector fitted with 20 &amp;micro;L stainless steel sample loop</td><td>Agilent</td><td>G1328C</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td></tr><tr><td>Ring Stand Mounting Bracket</td><td>Agilent</td><td>1400-3166</td><td></td></tr><tr><td>Agilent PLRP-S 300 &amp;Aring; 5&amp;nbsp;&amp;micro;m 4.6 x 250 mm (Analytical)</td><td>Agilent</td><td>PL1512-5501</td><td>http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td></tr><tr><td>A&amp;beta;42 or A&amp;beta;40 peptide</td><td></td><td></td><td>Synthesized in-house using a CEM liberty automated peptide synthesizer.</td></tr><tr><td>Ammonium Hydroxide (NH&lt;sub&gt;4&lt;/sub&gt;OH, 28% solution)</td><td>Fisher Scientific</td><td>A669-500</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific</td><td>A998-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Fisher Scientific</td><td>W5-4</td><td></td></tr><tr><td>Falcon 50 mL conical centrifuge tube</td><td>Fisher Scientific</td><td>14-954-49A</td><td></td></tr><tr><td>Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea</td><td>Sigma-Aldrich</td><td>Z227250</td><td></td></tr><tr><td>Normject 5&amp;nbsp;cc sterile syringe</td><td>Fisher Scientific</td><td>1481729</td><td></td></tr><tr><td>16 Gauge SS Needle</td><td>Rheodyne</td><td>3725-086</td><td></td></tr></tbody></table>

a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

The purification of the A&#946;42 peptide using a combination of the PLRP-S stationary phase and a high pH mobile phase results in the formation of a sharp, resolved peak for the A&#946; peptide at a retention time between 72 and 74 min (Figure 2C). Confirmation of the identity of the peak is done through direct injection mass spectrometry of the collected eluent. The eluent can be stored at -20 &#176;C in solution for up to 12 h. Longer periods of storage may result in oxidation of the protein. To isolate the purified peptide, the eluent is flash frozen in liquid nitrogen and lyophilized. Analytical HPLC of the purified peptide reveals a purity of &#62;95% (Figure 2D). The same procedure can be used to purify the A&#946;40 peptide (Figure 3A) without modification of the method. Analytical HPLC indicates a purity of A&#946;40 to be greater than 95% (Figure 3B).
<img alt="Figure 3" src="/files/ftp_upload/55482/55482fig3.jpg" /> 
Figure 3: Representative HPLC traces of A&#946;40. (A) Optimized preparative purification using the 25 x 300 mm2 polymer column, conditions are described in Table 1 located in sub-section 1.3 of the protocol description text. The peak corresponding to A&#946;40 is marked with an asterisk. (B) Analytical HPLC using the 4.6 x 250 mm2 polymer column, conditions: Buffer A: H2O with 20 mM NH4OH, buffer B: 80% MeCN / 20% H2O with 20 mM NH4OH, gradient: 5 to 50% buffer B over 30 min. Collection of the marked A&#946;40 peak in part A reveals a purity of &#62;95% as shown in part B. Mass spectrometry was used to determine the identity of the A&#946;40 peak. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55482/55482fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In order to confirm that the purified peptide can form oligomeric mixtures, we performed photo-induced crosslinking of unmodified proteins (PICUP) as previously described14,15 and were able to robustly observe oligomer formation.10

Watch this Scientific Journal Video about A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation at JoVE.com

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

a-tailored-hplc-purification-protocol-that-yields-high-purity-amyloid

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.8K Views.  University of California, Santa Cruz. Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer's disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Video: A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Neuroscience

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicine

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Chemistry

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biology

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.

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 ...

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Summary

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Chapters in this video

Preparation of Oligomeric β-amyloid_1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease

A11-positive β-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

Modeling Amyloid-β42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

Rapid Generation of Amyloid from Native Proteins In vitro

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