<html> 1. Deglicosilación enzimática <ol><li> Use tubos de PCR para minimizar la pérdida de agua por evaporación. Etiqueta de un conjunto de tubos de 1 a 7. </li><li> Descongele el tampón 10X G7, el buffer de la glicoproteína 10 veces desnaturalización, y el 10% de NP-40 y golpee suavemente los tubos para mezclar el contenido. Mantener a temperatura ambiente. </li><li> Colocar los viales que contienen enzimas en el hielo. Trate de minimizar el deshielo / congelación de los ciclos. </li><li> Disolver el contenido del vial hCGβ (150 mg) en 600 l de dH 2 O y mantener en hielo. </li><li> Prepare 1 ml de 1X buffer del G-7 mediante la dilución de las acciones 10 veces en dH 2 O. </li><li> Diluir 0,5 l de PNGase F en 25 l de amortiguación del G7 1X y mantener en hielo. </li><li> Prepare la mezcla exoglycosidase (mezcla de EG) mediante la combinación de 2 l cada uno de α-N-acetilgalactosaminidasa, fucosidasa α1-2, galactosidasa β1-3, y α1-3, 6 galactosidasa. </li><li> Establecer los tubos de PCR, como se indica: </li></ol>ak &quot;&gt; <table border="1"><tbody><tr><td> Tubo / de la muestra # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> Inhibidores de la glucoproteína 10X Buffer desnaturalización </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td></tr><tr><td> dH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> Tape los tubos, mezclar suavemente y colocar en eltermociclador, cierre la tapa y desnaturalizar las proteínas mediante la incubación de 10 minutos a 94 ° C, seguido de un 4 º C mantener (con tubos de PCR en un termociclador en gran medida evita la evaporación en las muestras de pequeño volumen). </li><li> Retire los tubos del termociclador y se centrifuga para eliminar la condensación visible. </li><li> Añadir los siguientes reactivos como se indica: </li></ol><table border="1"><tbody><tr><td> Muestra # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> 10% de NP-40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> Buffer 10X G7 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2,5 y mu; L </td><td> 2.5μl </td></tr><tr><td> PNGase F 1:50 dil. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Deglicosilación Mix </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> Mezcla de EG </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> dH 2 O </td><td> 10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> Vol total de reacción. </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l &lt;/ Td&gt; <td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td></tr></tbody></table><ol start="12"><li> Cerrar los tubos de PCR con tapas nuevas (deseche las que se utilizan, ya que no se ajustan correctamente después de un ciclo de incubación). </li><li> Mezcle los tubos golpeando suavemente 4 veces y luego girar el contenido hacia abajo. </li><li> Colocar los tubos en el termociclador e incubar a 37 ° C durante 4 horas y enfriar las muestras a 4 ° C. </li></ol> 2. SDS-PAGE de las muestras deglicosilada <ol><li> Prepare 130 l nuevo 3X tampón de carga SDS reducir mediante la adición de 4 l de 1,25 M de TDT. </li><li> Añadir 12,5 l de la preparada 3X tampón de carga SDS reduciendo a cada muestra. </li><li> Cerrar los tubos con tapones nuevos y golpee suavemente los tubos para mezclar. </li><li> Incubar los tubos en un termociclador a 94 ° C durante 5 minutos y dejar enfriar a 4 ° C. </li><li> Carga de 30 l de cada muestra y de 10 l de la proteína marcadora en un 10-20% de Tris-Glicina gel. Guarde el resto de la muestra de la parte 3.1. Cargar 10μl del marcador ColorPlus proteína prestained. </li><li> La electroforesis en gel a 130 voltios a temperatura ambiente hasta que el frente tinte en la parte inferior del gel. </li><li> Cuando el gel se haya terminado de ejecutarse, quitar el gel de los actores y el lugar en una pequeña caja de plástico con suficiente azul Coomasie mancha para cubrir el gel. </li><li> Manchar el gel durante 1 hora con agitación suave. </li><li> Lavar el gel tres veces durante 30 minutos en 50 ml de solución destain. </li><li> Grabar las imágenes utilizando un transiluminador de luz blanca o un escáner. Por otra parte, el gel se puede secar entre las hojas de papel de celofán en un marco. </li></ol> 3. Pro-Q Esmeralda 300 para la detección de proteínas glicosiladas en geles SDS-PAGE <ol><li> En paralelo con el gel de 2.1), carga el resto de las muestras en un 10-20% de Tris-Glicina gel. Carga de 10 y mu; L marcador de proteína ColorPlus prestained. </li><li> Mientras que el gel está ejecutando disolver el reactivo Pro-Q Esmeralda con DMF y preparar la revisión de valores, lavado y oxidantes soluciones para la Esmeralda Pro-Q 300 mancha siguiendo el manual de producto incluida con el kit. </li><li> Cuando la electroforesis, retire el gel de fundición y lo coloca en una caja de plástico. </li><li> Fijar el gel mediante la adición de 100 ml de la solución de Fix y dejarlo toda la noche a temperatura ambiente con agitación suave. </li><li> Lavar el gel con 100 ml de la solución de lavado de 10 a 20 minutos a temperatura ambiente con agitación gentil. Repetir el lavado con solución de lavado fresca. </li><li> Oxidar los hidratos de carbono mediante la incubación del gel con agitación suave durante 30 minutos en 25 ml de solución de oxidantes. </li><li> Lavar el gel como se describe en el paso 3.5. </li><li> Mientras que el gel es el lavado de preparar dulce Pro-Q Esmeralda 300 mancha añadiendo 500 l de la solución Pro-Q Esmeralda 300 reactivo disuelto en el paso 3.2 a 25 ml detinción de amortiguación en el kit. </li><li> Tinción del gel mediante la adición de 25 ml de la mancha preparado en el paso 3.8 e incubar en la oscuridad con agitación suave durante 90 a 120 minutos. </li><li> Repita los dos pasos de lavado descrito en el paso 3.5. </li><li> Grabar las imágenes con un transiluminador UV a 300nm. Use el marcador de 80 kDa, que está marcado con Pro-Q verde esmeralda, a la superposición de la imagen UV a una imagen de luz blanca del gel que muestra la escala prestained. </li><li> Compare las imágenes del gel Coomasie manchado en el paso 2.10 con el gel Pro-Q esmeralda teñidas de paso 3.11. </li></ol> 4. Los resultados representativos Los cambios en la migración de las proteínas después de deglicosilación enzimática se muestran en la Figura 2. Comparar la muestra de control (panel A, carril 1) con el tratamiento F PNGase (eliminación de N-glicanos, carril 2), y Mix deglicosilación (PNGase F, además de endo-y exoglicosidasas para eliminar O-glicanos, carril 3). Una reducción adicionalen el tamaño se ve después de digestión con glicosidasas adicional (calle 4). Además de un cambio en la masa, las bandas se agudizan como glicanos son removidos. Una banda ejecuta en el marcador de 17 kDa (asterisco) representa la totalidad deglicosilada hCGβ polipéptido (MW: 16 kDa). Otras bandas pudieran derivarse de deglicosilación incompleta o de las proteínas múltiples identificados en la muestra hCGβ (ver carril 1). Calles 5 a 7 (controles) muestran las bandas correspondientes a las glicosidasas. La tinción de la glicoproteína de verde esmeralda se muestra en el panel B. Este reactivo se oxida y manchas de todos los glicanos presentes en una molécula de proteína. Por lo tanto la intensidad de la señal disminuye a medida que hCGβ es enzimáticamente deglicosilada (calle 1 a calle 4). La señal residual en los carriles 3 y 4 indican la presencia de motivos glicanos, que son resistentes a las enzimas utilizadas. Las glicosidasas adicional que se utiliza en el carril 4 quitar unos pocos residuos adicionales de azúcar: la migración de las proteínas es el mismo, perouna ligera reducción en la intensidad de la tinción se puede ver. Fracciones resistentes al azúcar no estaban presentes en todas las especies de proteínas: algunas bandas no fueron detectados por el verde esmeralda (ausente en la imagen de los rayos UV, entre paréntesis), lo que indica que se deglicosilada ampliamente. Datos adicionales de apoyo a la conclusión de que hCGβ es heterogénea glicosilada. La banda inferior en la pista 2 (flecha) es débil en la imagen verde esmeralda, mientras que la banda superior en la pista 2 es brillante, lo que indica que los grupos de glicanos muchos todavía están presentes. Estos datos apoyan la conclusión de que hCGβ recombinantes expresadas en células de ratón contiene múltiples glicoformas 9. Estos glicoformas diferentes debido a la heterogeneidad inherente de la glicosilación en algunos polipéptidos no reciben un glicano en cada sitio de consenso y / o glicanos algunos se extienden, mientras que otros, incluso en la misma proteína que no lo son. <img alt="Figura 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> Figura 1.Los patrones típicos de glicosilación de las glicoproteínas secretadas o de la superficie celular. <img alt="Figura 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> Figura 2. Geles SDS-PAGE mostrando deglicosilación enzimática de hCGβ. El panel A muestra una coloración azul Coomasie mientras que el panel B se muestran los resultados de la Pro-Q Esmeralda 300 para la visualización de las proteínas glicosiladas. Número de la muestra: 1, el control hCGβ, 2, F PNGase digestión; 3, la digestión deglicosilación Mix, 4, Mix deglicosilación más exoglicosidasas digestión, las muestras de 5 a 7 son los controles de reactivos (O-Glyc, O-glucosidasa, GNA, β-N -acetilglucosaminidasa; fucosidasa Gal / FUC, β1-3 galactosidasa, α1-3, galactosidasa 6, y α1-2; GalNA, α-N-Acetylglalactosaminidase, Neu, la neuraminidasa; PNGase; F PNGase)

<ol>
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Los autores son empleados de New England Biolabs, que produce muchos de los reactivos utilizados en este artículo.

El método descrito aquí usando deglicosilación enzimática y SDS-PAGE puede proporcionar información valiosa sobre el estado de glicosilación de una proteína de interés, mientras que glicano reactivos específicos para facilitar la interpretación de los datos. Este protocolo está pensado para los estudios iniciales de la glicosilación de proteínas y es especialmente adecuado para secretora y las glicoproteínas de membrana de las células de mamíferos: las enzimas elegido en este caso concreto se eliminarán todos los N-glicanos, y N-glicanos o más y los azúcares más comunes la ampliación y que forman el núcleo de O-glicanos. Glicosidasas tienen la ventaja adicional de ser leve, en comparación con los métodos deglicosilación química, preservando la integridad de azúcares y de columna de la proteína. Para dilucidar la tasa de ocupación (que los aminoácidos son glicosilada), el grado de glicosilación, o para determinar la estructura fina de los glicanos, las técnicas más sofisticadas, tales como mespectrometría de culo, la cromatografía líquida o la RMN son obligatorios. Debido a su simplicidad, varios pasos en este protocolo se puede ajustar, sustituir y / o combinados para adaptarse a las distintas necesidades experimentales. Sin embargo, con el fin de obtener resultados que puedan interpretarse claramente que es importante para entender sus fortalezas y limitaciones. En primer lugar, la especificidad y la pureza de las glicosidasas son cruciales: sólo las enzimas bien caracterizadas evaluados para estar libres de las proteasas y otras actividades contaminantes deben ser utilizados. Desafortunadamente, no existe una definición estándar para la unidad de enzima glicosidasas, el usuario deberá determinar la sustitución adecuada de acuerdo a las especificaciones del fabricante. En segundo lugar, una cuidadosa selección de detección es necesario: a los reactivos de la proteína) tinción son útiles sólo si los resultados deglicosilación en un cambio significativo de la masa molecular. Como un resultado claro, como se muestra aquí no siempre se consigue. En otros casos, hemos visto mi anormalesintegración después de deglicosilación (lejos del peso molecular predicho, o la migración aún más lento). Este fenómeno no se entiende bien, pero se puede decir que cualquier cambio en la migración es evidencia de que la proteína se ha deglicosilada. B) la detección de azúcar con los anticuerpos presenta desafíos únicos que limitan su aplicabilidad. Ha sido muy difícil generar general anti-glicano anticuerpos, por lo general son planteadas contra un complejo objetivo de glicanos, lo que limita su uso. Por otra parte, varios anticuerpos monoclonales anti-glicanos anticuerpos mostrar reactividad cruzada no deseados 10. C) Las lectinas (proteínas con afinidad intrínseca de azúcar) son muy adecuadas para la detección de azúcar, además de que dan idea de la estructura de glicano. Sin embargo, no todos ellos tienen una especificidad estrecha, y muchos son sólo parcialmente caracterizado (lo que significa que podría tener afinidades desconocido). Como consecuencia, una tinción positiva lectina proporciona una indicación, no una prueba, de la presencia de un giVen azúcar. d) equipos de etiquetado de productos químicos (basado en la oxidación de peryodato de azúcares) son el método de elección para teñir todas las glicoproteínas, y por lo tanto muy adecuado para seguir deglicosilación. Cuando se procesa una muestra desconocida, es una buena práctica incluir los controles de la glicoproteína. Fetuina es un N disponible - y O-glicoproteína, la gonadotropina coriónica (dos subunidades) es también una buena opción. Albúmina de suero bovino (BSA) se puede utilizar como control negativo. Sin embargo, cabe señalar que algunas proteínas no glicosilada reaccionar ligeramente con la Esmeralda del Pro-Q 300, especialmente cuando se utiliza en altas concentraciones. No glicosilada estándares de peso molecular, tales como el marcador de la proteína prestained utilizados en este protocolo, tienen la ventaja de mostrar bandas nítidas. Sólo por casualidad, es una de estas proteínas (80 kDa) reactiva a Pro-Q Esmeralda 300. Por lo tanto, el usuario podría querer ejecutar una escala estándar de la glicoproteína en su lugar, como el caramelo de la caña de Invitrogen. Finalmente, simple en gel de detección de las glicoproteínas nucleocytosolic (que se modifican con una sola O-GlcNAc) es posible con el uso de un anticuerpo monoclonal, a lo largo de β-N-acetilglucosaminidasa para el control de especificidad 11. La descripción de esta técnica está más allá de la alcance de este artículo, pero debe ser mencionado como glicosidasas son herramientas útiles para el estudio de muchas otras glicoproteínas y glicoconjugados presentes en las células. Un tratamiento integral de todas las formas conocidas de la glicosilación se puede encontrar en la segunda edición de los &quot;Fundamentos de la Glicobiología&quot;, disponible gratuitamente en la biblioteca NCBI (ID estantería: NBK1908; PMID: 20301239) 12.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Β de gonadotropina coriónica humana </td><td> Sigma Aldrich </td><td> C6572 </td><td></td></tr><tr><td> Tubos para PCR </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> Termociclador PCR </td><td> Applied Biosystems </td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> P0704 </td><td> Se suministra con tampones </td></tr><tr><td> Mezclar proteínas deglicosilación </td><td> New England Biolabs </td><td> P6039 </td><td> Se suministra con tampones </td></tr><tr><td> α-N-Acetylglalactosaminidase </td><td> New England Biolabs </td><td> P0734 </td><td> Se suministra con buffer </td></tr><tr><td> α1-2 fucosidasa </td><td> New England Biolabs </td><td> P0724 </td><td> Se suministra con buffer </td></tr><tr><td> α1-3, 6 galactosidasa </td><td> New England Biolabs </td><td> P0731 </td><td> Se suministra con buffer </td></tr><tr><td> β1-3 galactosidasa </td><td> New England Biolabs </td><td> P0726 </td><td> Se suministra con buffer </td></tr><tr><td> Buffer 10X G7 (500 mM de fosfato sódico) </td><td> New England Biolabs </td><td> --- </td><td> Se suministra con PNGaseF o Mix Degl </td></tr><tr><td> Buffer 10X glicoproteína desnaturalización (5% SDS, 400 mM DTT) </td><td> New England Biolabs </td><td> --- </td><td> Se suministra con PNGaseF o Mix Degl </td></tr><tr><td> 10% de NP-40 </td><td> New England Biolabs </td><td> --- </td><td> Se suministra con PNGaseF o Mix Degl </td></tr><tr><td> 3X tampón de carga SDS (187,5 mM Tris-HCl pH 6,8, 6% SDS, 30% de glicerol, 0,03% bromophenol azul) </td><td> New England Biolabs </td><td> B7703 </td><td> Se suministra con 1,25 M de TDT </td></tr><tr><td> ColorPlus prestained marcador de la proteína </td><td> New England Biolabs </td><td> P7709 </td><td></td></tr><tr><td> 10-20% de Tris-Glicina Multigel </td><td> Cosmo Bio Co. </td><td> DCB-414893 </td><td></td></tr><tr><td> Cassette unidad de electroforesis </td><td> Cosmo Bio Co. </td><td> DCB-303111 </td><td></td></tr><tr><td> Electroforesis EPS de alimentación 301 </td><td> GE Healthcare </td><td> 18-1130-01 </td><td></td></tr><tr><td> Pro-Q Esmeralda 300 inhibidores de la glucoproteína manchas Kit </td><td> Invitrogen </td><td> P-21857 </td><td></td></tr><tr><td> Brilliant Blue R </td><td> Sigma Aldrich </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager HP System </td><td> Celular Biociencias </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glicosilación, la adición de azúcares covalentemente, es una de las principales modificaciones post-traduccionales de proteínas que pueden afectar significativamente a los procesos como la adhesión celular, el tráfico molecular, la limpieza, y la transducción de señales 1.4. En los eucariotas, las modificaciones de la glicosilación más comunes en la vía secretora son adiciones a los residuos de asparagina consenso (N-linked), o en residuos de serina o treonina (O-vinculado) (Figura 1). Iniciación de la síntesis de N-glicanos está altamente conservada en eucariotas, mientras que los productos finales pueden variar mucho entre las diferentes especies, los tejidos o las proteínas. Algunos glicanos quedan sin modificar (&quot;alta manosa N-glicanos&quot;) o procesados ​​en el aparato de Golgi (&quot;complejo de N-glicanos&quot;). Mayor diversidad se encuentra a O-glicanos, que comienzan con un común N-acetilgalactosamina (GalNAc) de residuos en las células animales, pero difieren en los organismos inferiores 1. ent &quot;&gt; El análisis detallado de la glicosilación de las proteínas es un campo en sí mismo y requiere muchos recursos y experiencia para ejecutar correctamente. Sin embargo, una variedad de enzimas disponibles que eliminar los azúcares (glicosidasas) hace posible tener una idea general del estado de glicosilación . una proteína en un laboratorio estándar Aquí se ilustra el uso de glicosidasas para el análisis de una glicoproteína de modelo. coriónica humana recombinante beta gonadotropina (hCGβ), que lleva dos N-glicanos y cuatro O-glicanos 5 La técnica requiere sólo simples instrumentación y consumibles típica, y puede ser adaptado fácilmente para el análisis de las muestras de la glicoproteína múltiples. Varias enzimas se pueden utilizar en paralelo al estudio de una glicoproteína. PNGase F es capaz de eliminar casi todos los tipos de N-glicanos ligados 6,7. De O-glicanos, no hay enzimas disponibles que puede romper un oligosacárido intacto de the esqueleto de la proteína. En cambio, O-glicanos se recortan por exoglicosidasas a un corazón corto, que luego se elimina fácilmente por O-glucosidasa. La mezcla de proteínas contiene deglicosilación PNGase F, O-glucosidasa, la neuraminidasa (sialidasa), galactosidasa β1-4 y β-N-acetilglucosaminidasa. Se utiliza para eliminar simultáneamente N-glicanos y algunos glúcidos O-8. Finalmente, la mezcla deglicosilación se complementó con una mezcla de exoglicosidasas otros (α-N-acetilgalactosaminidasa, α1-2 fucosidasa, α1-3, 6 galactosidasa, y galactosidasa β1-3), que ayudan a eliminar los monosacáridos otra resistencia que podrían estar presentes en ciertas O-glicanos. SDS-PAGE/Coomasie azul se utiliza para visualizar las diferencias en la migración de las proteínas antes y después del tratamiento glucosidasa. Además, un azúcar específico de método de tinción, ProQ Esmeralda-300, muestra de la señal disminuye a medida que los glicanos son successively eliminado. Este protocolo está diseñado para el análisis de pequeñas cantidades de glicoproteína (0,5 a 2 mg), aunque deglicosilación enzimática puede ser ampliado para dar cabida a grandes cantidades de proteínas, según sea necesario.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Usando glicosidasas específicas para eliminar los azúcares de las glicoproteínas seguido de SDS-PAGE es un método útil para detectar las modificaciones de glicanos de las muestras de proteínas y es una buena opción para los estudios de glicobiología inicial. Siguientes cambios deglicosilación pueden ser detectados como cambios en la movilidad en gel o por tinción con glicano reactivos sensibles.

Identificación y caracterización de la glicosilación de proteínas específicas de uso de endo-y exoglicosidasas

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Research

JoVE Journal

Biology

36.4K vistas.  New England Biolabs. Usando glicosidasas específicas para eliminar los azúcares de las glicoproteínas seguido de SDS-PAGE es un método útil para detectar las modificaciones de glicanos de las muestras de proteínas y es una buena opción para los estudios de glicobiología inicial. Siguientes cambios deglicosilación pueden ser detectados como cambios en la movilidad en gel o por tinción con glicano reactivos sensibles.

Video: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificación de los complejos de proteínas con la proteómica cuantitativa en S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Investigación

Biología

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Identificación de los socios interacción de proteínas en tándem de purificación de afinidad

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Anticuerpos, químicamente bloqueado Microarray de multiplexado de alto rendimiento de perfiles de glicosilación de proteínas específicas en muestras complejas

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Método para el aislamiento e identificación de mRNAs, microRNAs y componentes proteicos de ribonucleoprotein complejos de extractos celulares usando RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificación de los complejos de proteínas en<em&gt; Escherichia coli</em&gt; La purificación mediante afinidad del péptido secuencial en combinación con espectrometría de masas tándem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Generación, purificación y caracterización de especies invasivas en células de proteína DISC1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Pyrosequencing para la identificación microbiana y caracterización

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.