Este trabajo fue apoyado por becas y AG027818 AG030709 de NIH / NIA, 2005/2E de Larry L. Hillblom Foundation, IIRG-07-58334 de la Asociación de Alzheimer, y 07-65798 de California Departamento de Salud Pública.

1. Péptido de preparación <ol><li> Pesar ~ 100-200 mg de péptido liofilizado usando una microbalanza y la transferencia en la etiqueta, revestido con silicio, tubos de microcentrífuga de baja adsorbente. Aquí, nosotros usamos las secuencias humanas de Aβ40, Aß42, calcitonina y GRF. </li><li> Aquí, nosotros usamos los péptidos pre-tratados con 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) para obtener homogénea, global sin preparativos. Este paso es necesario porque preformados agregados inducir la agregación de las proteínas de rápida amiloidogénica, que se traducen en una mala reproducibilidad entre los experimentos 5. Otros métodos como la filtración y la SEC también se puede utilizar para obtener agregados sin los preparativos para picup 6. </li><li> Para el tratamiento de los péptidos con HFIP, pre-enfriar el contenedor de hielo dentro de HFIP en una campana de extracción usando la protección adecuada (HFIP es volátil y tóxico). De enfriamiento de un envase de 250 ml por lo general requiere 10-15 min. Añadir HFIP de pre-enfriado tubos que contienen péptidos liofilizados para obtener una concentración de péptido nominal de 0,5 mM. </li><li> Sonicar las soluciones de péptidos en un sonicador de baño de María durante 5 minutos a temperatura ambiente. </li><li> Vortex suavemente e incubar los tubos durante 30 minutos a temperatura ambiente. </li><li> Enfriar los tubos en hielo (durante 1 minuto), y dividir las soluciones en 10-50-l alícuotas de 0,6 ml etiquetados bajo adsorbente tubos. </li><li> Quitar HFIP por evaporación bajo una corriente ligera de nitrógeno, o dejar los tubos abiertos en la campana de humos durante la noche. Para la evaporación durante la noche, se colocan los tubos abiertos en un estante y los cubren con una hoja grande de Kimwipes para evitar el polvo y la contaminación de partículas. </li><li> Desecar el HFIP restantes en el vacío en un liofilizador, o un concentrador centrífugo durante 30 minutos, o en un desecador conectado a una toma de aspiración de 2 h. El producto final será una película de péptidos en la parte inferior de los tubos de microcentrífuga. Si bien exsiccated, los tubos se pueden almacenar hermético durante períodos prolongados (meses) a -20 o -80 ° C. </li></ol> 2. Solubilización de los péptidos HFIP tratados y foto entrecruzamiento <ol><li> Antes de solubilización de los péptidos HFIP tratados por reacciones de reticulación, es necesario preparar el cross-linking y reactivos de enfriamiento. </li><li> Pesar persulfato de amonio (APS, el Sr. 228,2 g / mol) y preparar una solución 20 mM de fosfato sódico 10 mM, pH 7,4. Mezclar con un agitar hasta que la solución es clara. </li><li> Prepare una solución mM de Tris (2,2-bipiridilo) dichlororuthenium (II) hexahidratado (RuBpy, el Sr. 748,63 g / mol) en fosfato sódico 10 mM, pH 7,4. Mezclar con un vórtice y verificar su completa disolución. Proteger el tubo de la luz con papel de aluminio. </li><li> Para el análisis de SDS-PAGE siguientes enlaces cruzados, un reactivo de enfriamiento conveniente es de 5% β-mercaptoetanol en 2 × SDS-PAGE muestra de amortiguación. Por otra parte, un M ditiotreitol (DTT, el Sr. 154,5 g / mol) en agua desionizada o un tampón adecuado se puede utilizar. </li><li> HFIP tratados con películas péptido se disolvió en NaOH diluido y luego tampón fosfato de sodio se agrega para obtener ~ 30 mM concentración de péptido. Añadir 60 mM NaOH seguido de agua destilada en el tubo que contiene la película péptido de tal manera que NaOH y el agua constituyen el 10 y el 45% del volumen final, respectivamente. Raspe la película de péptidos de las paredes en el interior del tubo de microcentrífuga con la punta, mezclar con la pipeta hacia arriba y abajo, y someter a ultrasonidos durante 5 minutos en un baño de ultrasonidos en baño María. </li><li> Añadir un 45% fosfato sódico 20 mM, pH 7,4, mezclar con la pipeta, y se centrifuga a 16.000 g durante 10 minutos. Dejar de lado alícuotas de descruzar vinculados péptidos se mezcla con el reactivo de enfriamiento en los controles negativos. </li><li> Ajuste el obturador de la cámara de retardo de 1 s. En tiempos de irradiación más alta, amplia reacciones de los radicales puede causar la degradación de proteínas. Cargar el obturador de la cámara. Tiempo de irradiación posible que tenga que ser optimizada al utilizar el método con una nueva proteína. </li><li> Una reacción típica picup se lleva a cabo en un volumen de reacción de 20 microlitros. Transferencia de 18 l de la solución de péptidos en una pared delgada, transparente, de 0,2 ml tubo de PCR. </li><li> Para la solución de péptido, agregue 1 RuBpy l seguido de un APS l y mezclar los reactivos con pipeta (la concentración final de RuBpy y APS en la mezcla de reacción de 0.05 y 1 mM, respectivamente). </li><li> Rápidamente coloque el tubo de reacción dentro de un frasco de vidrio de 1,8 ml. Coloque el vial en el interior del fuelle conectado por delante de la cámara. Coloque el protector del objetivo y pulse el disparador para que la muestra se irradia durante 1 s en el interior del fuelle. </li><li> Después de la irradiación de la muestra, rápidamente retire el vial del fuelle y el tubo de PCR de la ampolla. Rápidamente detener la reacción mediante la adición de una TDT l ó 10 l de amortiguación reduciendo página de muestra. Repita la reacción de cada alícuota de péptidos. </li><li> Las mezclas de reacción ya se puede congelar a -20 ° C para el almacenamiento de no más de 7 días o se mantienen en hielo antes de su análisis por SDS-PAGE y tinción con plata. </li></ol> 3. SDS-PAGE y tinción astilla-de los productos péptido reticulado <ol><li> Rutina de SDS-PAGE y tinción con plata-se realiza para visualizar reticulado péptidos. </li><li> Carga de 5 l de la mezcla de reacción por carril para obtener ~ 130 pmol de péptido por carril. </li><li> Incluyen cantidades similares de descruzar vinculados péptidos para la comparación. También incluye una escalera de proteínas estándar de aproximación visual de un peso molecular de las bandas de péptidos. </li><li> Ejecute el gel usando un aparato de electroforesis en gel estándar. Usamos la XCell SureLock Mini-célula del sistema de Invitrogen y realizar la tinción de plata de acuerdo a la publicación Invitrogen IM-1002, Novex Pre-Cast electroforesis en gel. </li></ol> Parte 4: Los resultados representativos (Figura 1) <img src="/files/ftp_upload/old/1071_2009_1_19_13/Result no demarcation.png" alt="Figura 1" /> Figura 1: plata manchada geles de poliacrilamida que muestra descruzar vinculados (-) y foto-reticulado (+) GRF, la calcitonina, Aβ40 y Aß42 SDS-PAGE y tinción de plata-análisis de picup generado Aβ40 oligómeros muestran intensidades de las bandas de aproximadamente similares de monómero a través de tetrámero, seguido por una fuerte disminución en la abundancia de oligómeros superiores 7. Descruzar vinculados Aβ40 migra con un consistente con la de monómero 7 señor. Aß42 muestra una distribución de oligómeros de tamaño distinto. Aß42 oligómeros comprenden grupos de 2-3 8. El primer grupo, a través de monómero trímero, muestra disminución de la intensidad con el fin de oligómeros en aumento. En el segundo grupo, una distribución de Gauss-como se observa entre tetrámero y heptamer, con un máximo de pentámero y hexámero. El tercer grupo, que no se muestra aquí, contiene oligómeros de Mr ~ 30.000-60.000 Da 8. Estos oligómeros superiores señor general, cuando se utilice SEC-LMW Aß42 aislados, pero no péptido preparado por filtración o tratamiento HFIP 6. Descruzar vinculados a Aß42 produce principalmente dos bandas, una banda de monómero y un amplio trimer / tetrámero banda, que es un artefacto inducido por SDS 5,9. La calcitonina se obtiene una distribución de tamaño de oligómero que difiere fuertemente de una disminución monótona exponencial de la región a través de monómero tetrámero, lo que sugiere la pre-existencia de dímeros, trímeros, tetrámeros y 7. El control de polipéptidos, GRF, produce una distribución de oligómeros monótona desde dímeros a través de hexamer de tal manera que las cantidades relativas de oligómeros aparente disminuye exponencialmente con el incremento de la masa molecular. En otros experimentos, los oligómeros superiores también pueden ser visualizados 7. El número exacto y la intensidad relativa de las bandas de oligómero pueden variar entre los experimentos en función de la concentración de proteínas reales, la cantidad de proteína cargada en el gel, y el tiempo empleado en la etapa de desarrollo en el protocolo de tinción de plata.

<ol>
	<li>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=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proc. Natl. Acad. Sci. 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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+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</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, 330-335 (2003).</li><li>Hepler, R. W., Grimm, K. M., Nahas, D. D., Breese, R., Dodson, E. C., Acton, P., Keller, P. M., Yeager, M., Wang, H., Shughrue, P., Kinney, G., Joyce, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+state+characterization+of+amyloid+&#946;-derived+diffusible+ligands.">Solution state characterization of amyloid &#946;-derived diffusible ligands.</a> Biochemistry (Mosc). 45, 15157-15167 (2006).</li><li>Bitan, G., 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=Rapid+photochemical+cross-linking&#8212;a+new+tool+for+studies+of+metastable,+amyloidogenic+protein+assemblies).">Rapid photochemical cross-linking&#8212;a new tool for studies of metastable, amyloidogenic protein assemblies).</a> Acc. Chem. Res. 37, 357-364 (2004).</li><li>Piening, N., Weber, P., Hogen, T., Beekes, M., Kretzschmar, H., Giese, A. <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+crosslinking+of+prion+protein+oligomers+and+prions.">Photo-induced crosslinking of prion protein oligomers and prions.</a> Amyloid. 13, 67-77 (2006).</li><li>Li, H. T., Lin, X. J., Xie, Y. Y., Hu, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+events+of+&#945;-synuclein+oligomerization+revealed+by+photo-induced+cross-linking.">The early events of &#945;-synuclein oligomerization revealed by photo-induced cross-linking.</a> Protein Pept. Lett. 13, 385-390 (2006).</li><li>Vollers, S. S., Teplow, D. B., 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=Determination+of+Peptide+oligomerization+state+using+rapid+photochemical+crosslinking.">Determination of Peptide oligomerization state using rapid photochemical crosslinking.</a> Methods Mol. Biol. 299, 11-18 (2005).</li></ol>

Picup fue desarrollado originalmente para estudiar complejos estables de proteínas 2. El método fue aplicado más tarde para el estudio cuantitativo de las asambleas metaestable proteína amiloide, incluyendo Aß 10, priones y las enfermedades asociadas a PrP Sc 11, y α-sinucleína 12. Los factores más importantes que deben considerarse al diseñar un experimento picup son la estequiometría reactivo, tiempo de irradiación, y el procedimiento de preparación de la muestra. Las dos primeras cu...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dolan-Jenner 200-W incandescent lamp</td>
<td>Other</td>
<td>Dolan-Jenner Industries</td>
<td>Model 170-D</td>
<td>The heat generated by the lamp does not affect samples for short incubation periods.</td>
</tr>
<tr>
<td>35-mm SLR camera body</td>
<td>Tool</td>
<td>Pentax</td>
<td>SP500 model</td>
<td>In our settings, a bellows is attached to the body of the camera to provide a convenient chamber for irradiation of the sample placed 10 cm away from the light source.</td>
</tr>
<tr>
<td>Clear, thin walled PCR tubes</td>
<td>Other</td>
<td>Eppendorf</td>
<td>951010006 supplied by Fisher L22-003-24</td>
<td>&nbsp;</td>
<tr>
<td>Glass vials (1.8 mL)</td>
<td>Other</td>
<td>Kimble Chromatography</td>
<td>60940A 2, supplied by Fisher 03-340-60</td>
<td>&nbsp;</td>
<tr>
<td>GRF</td>
<td>Reagent</td>
<td>Bachem</td>
<td>H-3695</td>
<td>&nbsp;</td>
<tr>
<td>HFIP</td>
<td>Reagent</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>A&beta;40 and A&beta;42</td>
<td>Reagent</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Calcitonin</td>
<td>Reagent</td>
<td>American Peptide</td>
<td>22-1-10</td>
<td>&nbsp;</td>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h. </td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7154-25 ML</td>
<td>Can be used when SDS-PAGE analysis is performed.</td>
</tr>
<tr>
<td>Novex Tricine SDS Sample Buffer (2x)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>LC1676</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10-20%)</td>
<td>Other</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine SDS Running Buffer (10x )</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC1675</td>
<td>&nbsp;</td>
<tr>
<td>Silver Express Staining Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6100</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

photo-induced cross-linking of unmodified proteins (picup) applied to amyloidogenic peptides

El ensamblaje de las proteínas amiloidogénica en oligómeros tóxicos es un acontecimiento fundamental en la patogénesis de las enfermedades de mal plegamiento de proteínas, como el Alzheimer, el Parkinson y las enfermedades de Huntington, esclerosis lateral amiotrófica hereditaria, y la diabetes tipo 2. Debido a la naturaleza metaestable de estos conjuntos de proteínas, es difícil evaluar su distribución de tamaño de oligómero cuantitativamente mediante los métodos clásicos, como la electroforesis, cromatografía de dispersión de la luz, fluorescencia, o dinámica. Oligómeros de proteínas amiloidogénica existen como mezclas metaestable, en el que los oligómeros se disocian en monómeros y asociarse en grandes asambleas de forma simultánea. Picup estabiliza la población oligómero por covalentes entrecruzamiento y cuando se combina con los métodos de fraccionamiento, como la electroforesis en gel de dodecil sulfato de sodio poliacrilamida (SDS-PAGE) o el tamaño de cromatografía de exclusión (SEC), ofrece picup instantáneas de la distribución de oligómeros de tamaño que existía antes de cruzar -linking. Por lo tanto, picup permite la visualización y el análisis cuantitativo de las poblaciones de proteínas metaestable y puede ser usado para monitorear el montaje y descifrar las relaciones entre las modificaciones de la secuencia y oligomerización<sup&gt; 1</sup&gt;. Mecánica, picup implica la foto-oxidación de Ru<sup&gt; 2 +</sup&gt; En un tris (bipiridilo) Ru (II) (RuBpy) a Ru<sup&gt; 3 +</sup&gt; Por irradiación con luz visible con la presencia de un aceptor de electrones. Ru<sup&gt; 3 +</sup&gt; Es un fuerte oxidante de un electrón capaz de abstraer un electrón de una molécula de proteína vecinos, generando una proteína radical<sup&gt; 1,2</sup&gt;. Los radicales son inestables y altamente reactivas y por lo tanto las especies desaparecen rápidamente a través de una variedad de reacciones intra e intermoleculares. Un radical puede utilizar la energía de un electrón no apareado para reaccionar con otro monómero proteína que forma un dímero radical, que posteriormente se pierde un átomo de hidrógeno y forma un estable, covalentemente ligado al dímero. El dímero puede reaccionar más a través de un mecanismo similar con monómeros o dímeros para formar oligómeros de orden superior. Ventajas de la relación picup a otros foto-químicos o métodos de entrecruzamiento<sup&gt; 3,4</sup&gt; Son cortas (≤ 1 s) la exposición de controles no destructivos de luz visible, sin necesidad de<i&gt; Pre facto</i&gt; Modificación de la secuencia nativa, y de longitud cero covalentes de entrecruzamiento. Además, picup permite el entrecruzamiento de las proteínas dentro de pH y temperatura, incluyendo los parámetros fisiológicos. Este sentido, demuestran la aplicación de picup de entrecruzamiento de tres proteínas amiloidogénica los 40 - y 42-residuo de proteína amiloide β-variantes (Aβ40 y Aß42), y la calcitonina, y una proteína de control, la hormona del crecimiento factor liberador (GRF).

Watch this Scientific Journal Video about Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides at JoVE.com

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Foto-inducido de entrecruzamiento de las proteínas sin modificar (picup) permite la caracterización de la distribución del tamaño de oligómero de mezclas de proteínas metaestable. Se demuestra la aplicación de picup a tres péptidos representante amiloidogénica los 40 - y 42-residuo forma de amiloide β-proteína, y la calcitonina, y un péptido de control de la hormona del crecimiento factor de liberación.

Foto-inducido el entrecruzamiento de las proteínas sin modificar (picup) aplicada a los péptidos amiloidogénicas

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

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Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

21.2K vistas.  University of California, Los Angeles. Foto-inducido de entrecruzamiento de las proteínas sin modificar (picup) permite la caracterización de la distribución del tamaño de oligómero de mezclas de proteínas metaestable. Se demuestra la aplicación de picup a tres péptidos representante amiloidogénica los 40 - y 42-residuo forma de amiloide β-proteína, y la calcitonina, y un péptido de control de la hormona del crecimiento factor de liberación.

Video: Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

Separación electroforética de las proteínas

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Investigación

Biología

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Tinción de proteínas en geles de

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Una lectina Método HPLC para enriquecer péptidos selectivamente glicosilada de muestras biológicas complejas

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

El aislamiento de lábil Multi-complejos de proteínas por In vivo Control Celular entrecruzamiento y Inmuno-cromatografía de afinidad magnética

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Microdisección láser aplicada a perfiles de expresión génica de subconjunto de las células de la Drosophila Ala Disco

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expresión cuantitativa y Purificación Aplicada a Recombinante Disulfide ricos en proteínas del veneno Producido en<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

La purificación no cromatográfico de polipéptidos recombinantes Elastina-como y sus fusiones con Péptidos y Proteínas de<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

El uso de MALDI-TOF espectrometría de masas y una base de datos personalizada para caracterizar bacterias indígenas a una cueva entorno único (Kartchner Cavernas, AZ, EE.UU.)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Un método para la selección de aptámeros Estructura de conmutación Aplicado a una de nanopartículas de oro ensayo colorimétrico

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Enriquecimiento de la Matriz Extracelular Las proteínas de los tejidos y la digestión en péptidos para el análisis de espectrometría de masas

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...