Name: a tailored hplc purification protocol that yields high-purity amyloid beta 42 and amyloid beta 40 peptides, capable of oligomer formation
Uploaded: 2017-03-27T14:00:00
Description: 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

Los autores desean agradecer a Agilent para su asistencia técnica. Kate Markham y Rafael Palomino se acreditan por su ayuda inicial para la síntesis y purificación del péptido Aß y el Dr. Hsiau-Wei Lee se dio las gracias por su ayuda en la preparación de la Figura 1 del manuscrito.

1. La HPLC preparativa purificación del Aß40 o Aß42 Péptido <ol><li> Preparar los siguientes tampones para la purificación por HPLC. <ol><li> Preparar el tampón A (20 mM NH 4 OH) mediante la adición de 1,3 ml de NH4OH (solución al 28%) a 1000 ml de agua ultrapura. </li><li> Preparar el tampón B (80% de acetonitrilo con 20 mM NH 4 OH) mediante la adición de 1,3 ml de NH 4 OH (solución al 28%) a una solución de 800 ml de acetonitrilo de grado HPLC ...

<ol>
	<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. 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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. 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La purificación por HPLC del péptido Aß es altamente dependiente de la elección tanto de la fase estacionaria empleada en la purificación y la fase móvil elegido para eluir el péptido. El punto isoeléctrico bajo del péptido y la alta propensión a la agregación render condiciones cromatográficas tradicionales para la separación de proteínas hidrófobas (C4 o fase estacionaria C8 junto con un eluyente móvil ácida) un reto, con el péptido Aß eluyendo como un prolongado amplio, no resuelta pico (Fi...

La enfermedad de Alzheimer es un trastorno neurodegenerativo que los efectos más de 35 millones de personas en todo el mundo. 1 Implicado fuertemente en la aparición y desarrollo de la enfermedad, es la agregación altamente propensa, péptido amiloide beta hidrófobo (Aß). 2 Aß se extiende de 36 a 43 aminoácidos de longitud, sin embargo, se cree que la variante de ácido 42-amino, amiloide beta 42 (Aß42), es la forma más tóxica de la proteína. 3 Esto se debe en su mayor parte a la capacidad de Aß42 para formar fácilmente difusibles, especies oligoméricas que se cree que son entidades particularmente neurotóxicos. 4 Con el fin de mejorar nuestra comprensión del péptido Aß, es esencial para obtener de forma rutinaria material de alta pureza. La presencia de trazas de impurezas se ha demostrado que alterar dramáticamente las propiedades de agregación de propensión del péptido. 5 Tradicionalmente, la cromatografía (HPLC) de separación de líquidos de alto rendimiento de péptidos hidrófobos tales como Aß se ha realizado mediante el uso de una combinación de C 4 o C 8 fases estacionarias a base de sílice y una fase móvil ácida. 6 Sin embargo, tales condiciones pueden presentar un reto a la purificación del péptido. El punto isoeléctrico bajo del péptido Aß (pI de aproximadamente 5,5) 7 significa que bajo condiciones ácidas, la agregación de péptido se aumenta y como resultado picos anchos de HPLC, no resueltos que a menudo son difíciles de aislar se producen (Figura 2A). Además, tales picos anchos a menudo contienen impurezas que pueden afectar el perfil de agregación del péptido, y comúnmente requieren las siguientes rondas de purificación que pueden afectar dramáticamente la cantidad de péptido producido. El poli (estireno / divinilbenceno) fase estacionaria, PLRP-S, representa un medio alternativo de tratamientpéptidos hidrofóbicos ying. La fase estacionaria se ha empleado en la purificación de un número de diferentes proteínas y ácidos ribonucleico mensajero (ARNm). 8, 9 La fase estacionaria PLRP-S no requiere ligando alquilo adicional para la separación de fase inversa, y más importante es químicamente estable a pH alto que conduce a la desagregación del péptido. 7 Aquí, se presenta un protocolo de purificación por HPLC a medida que los rendimientos de alta pureza beta amiloide 42 (? 42) y beta amiloides 40 (AP40).

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		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent 1260 Infinity II quarternary pump</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">G7111B</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-pumps-vacuum-degassers/1260-infinity-ii-quaternary-pump</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent 1260 Infinity II Dual variable wavelength detector</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">G7114A</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-detectors/1260-infinity-ii-variable-wavelength-detector</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:207px;">Agilent 1260 Infinity II Manual Injector fitted with 10 mL stainless steel sample loop</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">0101-1232</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:207px;">Agilent 1260 Infinity II Manual Injector fitted with 20 &#181;L stainless steel sample loop</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">G1328C</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-injection-systems/1260-infinity-ii-manual-injector</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">Ring Stand Mounting Bracket</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">1400-3166</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:207px;">PEEK Tubing Blue (1/32&#34; outer diameter &#1093; 0.010&#34; internal diameter)</td>
			<td style="width:85px;">Thermo Scientific</td>
			<td style="width:179px;">03-050-399</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:207px;">Agilent PLRP-S 300&#197; 8&#181;m 25 &#1093; 300 mm column (Preparative)</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">PL1212-6801</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent PLRP-S 300&#197; 8&#181;m 7.5 &#1093; 300 mm (Semi-Preparative)</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">PL1112-6801</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Agilent PLRP-S 300&#197; 5 &#181;m 4.6 x 250 mm (Analytical)</td>
			<td style="width:85px;">Agilent</td>
			<td style="width:179px;">PL1512-5501</td>
			<td style="width:739px;">http://www.agilent.com/en-us/products/liquid-chromatography/lc-columns/biomolecule-separations/plrp-s-for-biomolecules#features</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">A&#946;42 or A&#946;40 peptide</td>
			<td style="width:85px;"></td>
			<td style="width:179px;"></td>
			<td style="width:739px;">Synthesized in-house using a CEM liberty automated peptide synthesizer.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:207px;">Ammonium Hydroxide (NH4OH, 28% solution)</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">A669-500</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Acetonitrile</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">A998-4</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">HPLC grade water</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">W5-4</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Falcon 50 ml conical centrifuge tube</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td style="width:179px;">14-954-49A</td>
			<td style="width:739px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Supelco PEEK Fitting One-piece fingertight, pkg of 5 ea</td>
			<td style="width:85px;">Sigma-Aldrich</td>
			<td style="width:179px;">Z227250</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:207px;">Normject 5cc sterile syringe</td>
			<td style="width:85px;">Fisher Scientific</td>
			<td>1481729</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:207px;">16 Gauge SS Needle</td>
			<td style="width:85px;">Rheodyne</td>
			<td style="width:179px;">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.

La purificación del péptido Aß42 usando una combinación de la fase estacionaria PLRP-S y un pH alto resultados de fase móvil en la formación de un pico agudo, resuelto para el péptido Aß en un tiempo de retención entre 72 y 74 min (Figura 2C). La confirmación de la identidad del pico se realiza a través de inyección directa de espectrometría de masas del eluyente recogido. El eluyente se puede almacenar a -20 ° C en solución para un máximo de 12 h. Perío...

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

Aquí nos presenta un protocolo de purificación por HPLC a medida que los rendimientos de alta pureza beta amiloide 42 (? 42) y beta amiloides 40 (AP40), apto para la formación de oligómeros. El amiloide beta es un péptido altamente propensos agregación, hidrófoba implicada en la enfermedad de Alzheimer. La naturaleza del péptido amiloidogénico hace su purificación un desafío.

Un protocolo de purificación HPLC Tailored que los rendimientos de alta pureza beta amiloide 42 y 40 péptidos beta amiloide, capaz de Formación de oligómeros

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