Este trabajo fue financiado con fondos de la Fundación Canadiense para la Innovación, Genoma Canadá, el Instituto de Genómica de Ontario, Ontario el Ministerio de Innovación, y los Institutos Canadienses de Investigación en Salud de subvención a JG y AE E. Rojo-expresión DY330 coli cepa fue una especie de regalo de Donald L. Corte (National Cancer Institute, Frederick, MD).

1. Construcción de Genes específicos de SPA-tagging en E. DY330 coli cepa <ol><li> El plásmido pJL148 que abarca la secuencia de ADN SPA-tag y el antibiótico kanamicina casete marcador de resistencia (Kan R) se utiliza como una plantilla en la reacción en cadena de polimerasa (PCR) 7. A 45 nt de genes específicos de cebador directo, que se encuentra inmediatamente aguas arriba del codón de parada del gen diana en el marco con un 27 bp (&#39;5&#39;-AGCTGGAGGATCCATGGAAAAGAGAAG -3) tag cebador específico, y un 45 nt de genes específicos de cebador inverso, situado inmediatamente abajo del codón de parada del gen diana en el marco con un pb 27 (5&#39;-GGCCCCATATGAATATCCTCCTTAGTT -3 &#39;) cebador inverso etiqueta específica, se usan para amplificar el SPA-tag y casete Kan R de la pJL148 utilizando las siguientes condiciones de ciclo de PCR: 94 ° C durante 5 min, 30 ciclos de 94 ° C por 1 min, 55 ° C durante 1 min, 68 ° C durante 2 min 10 seg, seguido de 68 ° C durante 10 min. Estas secuencias amplificadas servir uns sustratos para la ectópica introducido λ-Rojo maquinaria de recombinación homóloga (véase a continuación). </li><li> El producto de PCR se purifica utilizando un kit de purificación de PCR QIAquick para eliminar las sales que pueden interferir con la electroporación. El producto de PCR purificado es posteriormente dirigida a integrar en el extremo 3 &#39;(inmediatamente aguas arriba del codón de parada nativa) de un gen específico en DY330 cepa en la que se expresa el λ-Rojo maquinaria de recombinación 10. </li><li> El DY330 λ-Red cepa que expresa se cultivaron durante la noche en 2 ml de medio Luria-Bertani (LB) a 32 ° C con agitación a 180 rpm. 1 ml del cultivo durante toda la noche posteriormente se inocularon en 70 ml de medio LB fresco en 500 ml matraz cónico. El inóculo se cultivaron a 32 ° C con agitación a 180 rpm hasta que la DO 600 alcanza ~ 0,8. </li><li> El cultivo se transfirió a un nuevo matraz de 250 ml cónico, donde las células son inducidas mediante la incubación el matraz en un baño de agua a 42 ° C agitando suavemente en180 rpm durante 15 min. Inmediatamente después de la inducción, el matraz se incuba en un baño de hielo suspensión en agua durante al menos 30 min con agitación. </li><li> El cultivo enfriado con hielo se transfiere inmediatamente en pre-refrigerada 50 ml tubo de polipropileno y se sometieron a centrifugación a 3.993 xg durante 6 min a 4 ° C. El sedimento celular se resuspendió en 50 ml de helado de agua estéril y se centrifugaron de nuevo a 3.993 xg durante 6 min a 4 ° C. El sedimento de células se resuspende en 1 ml de agua fría y se transfirió a un tubo de 1,5 ml Effendorf, y se centrifugó a velocidad máxima durante 20 segundos a 4 ° C. Después de dos o tres etapas de lavado con agua fría de hielo, el precipitado celular se resuspendió finalmente en 700 l de agua enfriada con hielo estéril, resultando en electro-competentes células. </li><li> A través de la electroporación, una l de la amplicón purificado se introduce en 40 l de las células electro-competentes. Las células se someten a electroporación en un II Bio-Rad GenePulser con los siguientes ajustes: 2,5 kV, 25 mF con pulsocontrolador de 200 Ω. Después de la recombinación homóloga y la integración, los transformantes que recombinados con éxito la etiqueta / casete en el cromosoma se seleccionan basándose en la resistencia a Kan (Figura 1A). Varios transformantes se seleccionan para el Western Blot para verificar la correcta generación (es decir, la señal positiva) de SPA-etiquetados proteínas de fusión con anticuerpos anti-FLAG M2 que es selectivo contra los epítopos bandera de la SPA-tag. </li><li> Confirmado carboxi terminal SPA-tag cepa de fusión se cultivaron en una gran escala para aislar complejos proteína soluble a partir de células cosechadas utilizando el protocolo de purificación de SPA. El esquema de los pasos implicados en el procedimiento de purificación por afinidad etiqueta se muestra en la Figura 1B. </li></ol> 2. El cultivo y la sonicación <ol><li> Inocular 100 l de una E. SPA-etiquetado Imágenes coli glicerol en 50 ml de caldo Terrific (TB) medio líquido suplementado con 50 l de 25 mg / ml of Kan solución en un matraz cónico de 250 ml. Mantener el cultivo durante la noche a 32 ° C. Nota: Puesto que el casete λ temperatura inducible Red en la cepa DY330 está bajo el control de un represor sensible a la temperatura 10, la cepa SPA-etiquetados E. coli se hace crecer a 32 ° C. </li><li> Transferir 10 ml del cultivo durante la noche en 990 ml de TB fresco suplementado con 25 g / ml de Kan en un matraz de 4 litros. El cultivo se hace crecer a 32 ° C con agitación constante a 250 rpm, durante 5 a 6 horas, hasta que la DO 600 alcanza a ~ 2 a 3. </li><li> Transferir el 1 litro E. coli SPA-etiqueta cultura para limpiar botellas de centrifugación y centrifugar las células a 4 º C en Beckman J6-HC centrífuga @ 3993 xg durante 15 min. </li><li> El sobrenadante se elimina de las botellas de centrifugación. Resuspender el E. gránulos coli células con 25 ml de tampón de sonicación. Asegúrese de mantener las botellas de centrifugación sobre hielo en todo momento. </li><li> El precipitado resuspendido setransferido a 50 ml de polipropileno tubo Falcon y se congelan con nitrógeno líquido. Las células congeladas se almacenan a -80 ° C para uso a largo plazo. </li><li> Antes de la sonicación, las células congeladas descongelar completamente, manteniendo los gránulos en hielo. Las muestras descongeladas se transfirió a un vaso de acero inoxidable estéril colocó en hielo durante la sonicación. La sonda se sumerge en la muestra y se sonicó durante 3 min. Un adicional de 2 min se deja enfriar la muestra del sobrecalentamiento. </li><li> El lisado celular sonicado se transfiere en el tubo de centrifugación pre-refrigerada estéril, y se sometió a centrifugación a 4 ° C en una centrífuga Beckman usando un rotor JA-17 @ 35.267 xg (16.000 rpm) durante 15 min dos veces. Nota: En el caso de la purificación de proteínas de membrana, el lisado celular sonicado se centrifuga sólo una vez durante 15 min, ya que no sabe en qué fracción de las proteínas de membrana son, es decir, ya sea en el sedimento o en la fracción sobrenadante. Así que para evitar pérdida excesiva de memproteínas de membrana, hacemos girar las células durante 15 min a centrifugación a alta velocidad y el sobrenadante se procesa posteriormente para la purificación (ver pasos abajo) donde el 1% se usa un detergente para solubilizar las proteínas de la membrana. </li><li> El sobrenadante de la centrifugación en tubo se transfiere cuidadosamente a un tubo de 50 ml de polipropileno Falcon y se congelaron con nitrógeno líquido. El extracto sonicado de células congeladas se almacena durante un período máximo de 6 meses a -80 ° C para su uso futuro. </li></ol> 3. Purificación por afinidad <ol><li> Antes de su uso, 100 l de anti-FLAG M2 perlas se lavan por 10 volúmenes de tampón de AFC (30 mM Tris-HCl, 150 mM NaCl, 0,1% de detergente, y 0.1-0.5 mM TCEP [tris (2-carboxietil) fosfina - ácido clorhídrico]) sin el detergente y el agente reductor de TCEP (tris (2-carboxietil) fosfina). </li><li> El extracto congelado, sonicado celular se descongela colocando el tubo en agua fría. El extracto de células descongeladas se incubaron con 3 l de benzonase nucleasa durante 30 min a 4 ° C. A esta mezcla, agregar un detergente no iónico Triton X-100 (concentración final del detergente debe ser 0,1%) y 200 l de suspensiones anti-FLAG M2 perlas de agarosa. Nota: Para todas las purificaciones de proteína soluble, 0,1% de Triton X-100 se utiliza para minimizar la adsorción no específica, mientras que para la purificación de una proteína de membrana, diferentes suaves detergentes no iónicos, tales como C12E8 (éter de octaetilenglicol dodecil glicol), DDM ( n-dodecil-β-D-maltósido) y maltosa-neopentilglicol (MNG) se puede utilizar en una concentración de detergente 1% para mejorar la solubilización. Ya que los detergentes diferentes tienen diferentes eficiencia de solubilización de proteínas de membrana, se recomienda llevar a cabo purificaciones independientes de proteínas utilizando más de un detergente. </li><li> El contenido se mezcla suavemente haciendo girar el tubo durante 3 horas a 4 ° C usando un agitador de LabQuake. Después de 3 horas de rotación, el tubo se centrifugó a 1.700 xg durante 6 min. El sobrenadante es entonces removed cuidadosamente, tanto como sea posible, sin perturbar el sedimento suelto perla. </li><li> El sedimento se resuspende en el sobrenadante restante y luego se transfiere a un 0,8 x 4 cm de columna Bio-Rad polipropileno prep. Asegúrese de quitar los tapones de salida inferior de la columna, para permitir que los eluatos para drenar por gravedad. Lavar la columna con 5 veces el paso de escisión TEV. </li><li> Después de drenar los eluatos lavados, la salida del fondo de la columna está cerrado. Para la misma columna, la escisión se lleva a cabo mediante la adición de ~ 5 a 10 l (50 unidades) de la proteasa TEV y 400 l de 1X tampón de AFC en la columna. Asegúrese de cerrar la parte superior de la columna con una tapa. La columna que contiene las perlas se gira suavemente durante la noche a 4 ° C durante la noche usando un agitador LabQuake. </li><li> Quitar el tapón en la parte superior y el tapón de salida en la parte inferior de la columna, y la fuga de los eluatos recuperados después de la escisión TEV en una columna reciente que contenía 200 l de suspensiones calmodulina-sepharose bolas, que se lava por 10volúmenes de tampón de unión a calmodulina. </li><li> Tanto la parte superior y la parte inferior de la columna se sujetan firmemente, y el contenido de la columna se mezcló suavemente por rotación durante 3 horas a 4 ° C usando un agitador de LabQuake. </li><li> Después de 3 horas de rotación, el eluato se drena mediante la eliminación de la tapa superior y el tapón de fondo de la columna. La columna se lava cuatro veces con 200 l de 1X tampón de unión a calmodulina seguido por un lavado riguroso con 400 l de tampón de lavado calmodulina. </li><li> La proteína unida se eluyó en cuatro fracciones de 50 l en un tubo Eppendorf usando 1X tampón de elución calmodulina. La fracción eluida se distribuye en dos tubos Eppendorf limpios en volúmenes iguales. Tanto los tubos se secan posteriormente usando un vacío de velocidad. </li><li> El eluato se secó de un tubo se utiliza para el funcionamiento de un gel de tinción de plata, mientras que la otra se almacena en -80 ° C, antes de usar la espectrometría de masas. </li></ol> 4. Silver Staining <ol><li> Para la plata staining, la mitad del volumen de 3X SDS (dodecil sulfato sódico) tampón de muestra se añade a 50 l de eluato se secó. Después de hervir la mezcla durante 5 min, las muestras se cargaron en un gel de poliacrilamida SDS. </li><li> Después de que el gel ha finalizado la ejecución, que se coloca cuidadosamente en la solución de fijación (50% de metanol y 10% de ácido acético) y se agitó suavemente en un agitador rotatorio durante 20 min. El gel después se enjuaga con 20% de etanol durante 10 min. </li><li> El gel fijo se lava dos veces a fondo con 500 ml de agua doblemente destilada durante 10 minutos para asegurar fondo uniforme bajo. </li><li> Después el gel se agitó suavemente en 500 ml de tiosulfato de sodio durante 1 min, seguido por dos etapas de lavado con agua destilada durante 20 segundos. </li><li> El agua se elimina, y el gel se incuba en 200 ml de nitrato de plata 0,1% durante 30 min. Después de ese tiempo, el nitrato de plata se retira y se lava el gel con agua destilada durante 20 segundos para eliminar el exceso de nitrato de plata. </li><li> El gel es finalmente a mano agita en 75 mlde la solución de revelado. Cuando la intensidad deseada, la solución de revelado se descarta y 80 ml de ácido acético se añade para detener la reacción. Asegúrese de incubar el gel en ácido acético durante un mínimo de 20 min para la correcta visualización de bandas de gel. </li></ol> 5. La proteólisis y preparación de muestras para espectrometría de masas <ol><li> Para la muestra seca, se añaden 50 l de tampón de digestión y 0,9 l de 100 mM TCEP-HCl (tris (2-carboxietil) fosfina - ácido clorhídrico) y se incuba la mezcla durante 45 min a temperatura ambiente para la etapa de reducción. A continuación, 1 l de yodoacetamida 500 mM y se incuba en la oscuridad durante 40 min adicionales para permitir la alquilación de la muestra. </li><li> Después de la segunda ronda de la incubación, añadir 1 g de tripsina inmovilizada a la mezcla e incubar bien a 37 ° C durante 5 horas o durante la noche a temperatura ambiente. Asegúrese de detener la reacción mediante la adición de 1 l de ácido acético. </li><li> Humedecer previamente el Millipore Zip-Tip punta de pipeta de aspiración 10 l de la solución humectante y equilibrado. Vierta la solución a los residuos. Posteriormente aspirar 10 l de la solución de lavado y dispensar la solución a los residuos. Repita este paso dos veces. </li><li> Para una unión eficaz de la mezcla de péptidos a la punta, pipeta mezclar la mezcla de péptidos 20 veces. La mezcla de péptidos que se adhería a la punta es lavado por aspiración y dispensación de la solución de lavado. Repita este procedimiento dos veces para una unión eficiente de la mezcla de péptidos a la punta. </li><li> Con la punta que contiene el péptido unido, aspire 10 l de la solución humectante y la equilibración, y se distribuye en un tubo eppendorf limpio. Repita este paso dos veces. Después de secar las muestras eluidas en vacío velocidad, las muestras pueden ser analizadas por espectrometría de masas inmediatamente o se almacenaron a -80 ° C antes de su uso. </li></ol> 6. Identificación de proteínas por LTQ Velos Orbitrap Espectrómetro de Masas Thcomponentes electrónicos de polipéptidos de los complejos aislados se identifican utilizando una Orbitrap LTQ Velos híbrido espectrómetro de masas en tándem. El Orbitrap tiene una potencia excepcional resolver (&gt; 60.000 Máximo completa Media Ancho, o FWHM) y exactitud de la masa (&lt;2 ppm) que minimiza MS / MS de muestreo de irrelevantes contaminantes de fondo no específicas detectadas en purificaciones de control, mientras que la velocidad alta Velos trampa de iones componente puede detectar y péptidos fragmento baja abundancia utilizando tanto la transferencia de electrones y la disociación inducida por colisión modos de disociación. Partidos de alta confianza entre resultante MS / MS espectros de referencia se asignan a E. secuencias de proteínas coli utilizando el algoritmo de búsqueda de base de datos como SEQUEST y cada secuencia correspondiente evaluados durante un algoritmo de probabilidad como STATQUEST 11. El número total de espectros, la singularidad y la secuencia de péptidos contaminantes comunes fondo detectados en el control negativo (es decir. Simulado) purificaciones son consideradas para lograr la máxima empírico falso disdescubrimiento tasa. Los siguientes pasos se realizan para la identificación de proteínas: <ol><li> Las columnas micro-se embalan con ~ 10 cm de 3 m Luna C-18 de resina y están interconectados a una fuente de iones de nanoelectropulverización Proxeon que se coloca en línea con el instrumento Orbitrap. </li><li> Un Proxeon flujo binario nano HPLC bomba se utiliza para proporcionar una velocidad de punta de flujo estable de ~ 300 nl min -1 durante las separaciones de péptidos. </li><li> Para conseguir la elución de péptidos, un gradiente de tampón orgánico se establece de acuerdo a la complejidad de la muestra. Por ejemplo, el siguiente gradiente normalmente se configura para E. muestras coli SPA: disolvente B se aumentó de 2% a 6% en 1 min, a 24% en 38 min, a 100% en los próximos 4 min, se mantuvo a 100% durante 1 min, y luego se redujo a 2% en 1 min, y final de mantener a 2% durante 15 min. La fase móvil disolvente A tiene 95% HPLC gradiente de agua y 5% de ACN con 0,1% de ácido fórmico, mientras que el disolvente B tiene 5% HPLC gradiente de agua y 95% de ACN con fórmico 0,1% acid. La velocidad de flujo en la punta de la aguja se establece en -1 300 nl min durante 60 min. </li><li> A medida que los ciclos del espectrómetro de masas ejecutar a través de un ciclo completo de masas en 60.000 resoluciones, 10 concomitantes tándem escaneos masivos de fragmentación son recogidos por los iones precursores más intensos. Exclusión dinámico, selección masal monoisotópico, y en tiempo real los datos que dependen de características del instrumento de adquisición han sido habilitadas. </li><li> Los espectros se buscan utilizando un algoritmo de búsqueda de bases de datos tales como SEQUEST contra una base de datos de E. coli secuencias de proteínas, y el resultado es estadísticamente filtrada utilizando un algoritmo de probabilidad como STAQUEST 11 para garantizar una baja tasa de falsos descubrimiento. Sólo los candidatos de confianza alto (99% de probabilidad) se seleccionan, mientras que los partidos que muestra más de 10 ppm de masa de error en comparación con la masa del péptido teórico se eliminaron de otros estudios. </li></ol>

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No hay conflictos de interés declarado.

Un aspecto clave del enfoque basado en SPA APMS descrito aquí es que el marcado se realiza en el contexto cromosómico natural, garantizando así la regulación de genes normales se mantiene (es decir. Cebo promotor nativo conserva, por lo tanto, los niveles de expresión no es perturbado) y nativo de manera estable asociada a complejos de proteínas se recuperan casi a los niveles endógenos. Cuestiones operón polaridad también se evitan mediante la inclusión de un promotor orientada hacia fuera en el marc...

<table border="1">
	<tbody>
		<tr>
			<td>Materials</td>
			<td>Vendor</td>
			<td>and Catalog Numbers</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>I. Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Bioshop</td>
			<td>#KAN201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>#AMP201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Terrific-Broth medium</td>
		</tr>
		<tr>
			<td>Bio-Tryptone</td>
			<td>Bioshop</td>
			<td>#TRP 402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bioshop</td>
			<td>#YEX 555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bioshop</td>
			<td>#GLY 002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Bioshop</td>
			<td>#PPM 302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Bioshop</td>
			<td>#PPM 303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. Bacterial Strain and Plasmid</td>
		</tr>
		<tr>
			<td>DY330</td>
			<td>&nbsp;</td>
			<td>Yu et al. (2000)10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pJL148</td>
			<td>&nbsp;</td>
			<td>Zeghouf et al. (2004)7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. PCR and Electrophoresis Reagents</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 X PCR buffer</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 mM dNTPs</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 mM MgCl2</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td># AGA002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Loading dye</td>
			<td>NEB</td>
			<td>#B7021S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Bioshop</td>
			<td># ETB444</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Thermo Scientific</td>
			<td>#28355</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Bioshop</td>
			<td>#TRS001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Bioshop</td>
			<td>#BOR001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Sigma</td>
			<td># E6768</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNA ladder</td>
			<td>NEB</td>
			<td>#N3232L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. Plasmid isolation and Clean-up Kits</td>
		</tr>
		<tr>
			<td>Plasmid Midi kit</td>
			<td>Qiagen</td>
			<td>#12143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>#28104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. PCR and Transformation Equipments</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>BioRad</td>
			<td>iCycler</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis</td>
			<td>BioRad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-Rad</td>
			<td>GenePulser II</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.2 cm electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>42 &deg;C water bath shaker</td>
			<td>&nbsp;</td>
			<td>Innova 3100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beckman Coulter TJ-25 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>TS-5.1-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C Shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C large Shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C plate incubator</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>7. Electrophoresis and Western blotting</td>
		</tr>
		<tr>
			<td>Acrylamide monomer, N,N'- methylenebis-acrylamide</td>
			<td>Bio-Rad</td>
			<td>#161-0125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bioshop</td>
			<td># AMP001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>n-butanol</td>
			<td>Sigma</td>
			<td># B7906</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bioshop</td>
			<td>#TEM001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Whatman No. 1 filter paper</td>
			<td>Fischer Scientific</td>
			<td>#09-806A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mini protean 3 cell</td>
			<td>Bio-Rad</td>
			<td>#165-3301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>iBlot gel transfer device</td>
			<td>Invitrogen</td>
			<td>#IB1001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>Bio-Rad</td>
			<td>#162-0115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Flag M2 antibody</td>
			<td>Sigma</td>
			<td>#F3165</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Amersham</td>
			<td>#NA931V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pre-stained protein molecular weight standards</td>
			<td>Bio-Rad</td>
			<td>#161-0363</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chemiluminescence reagent</td>
			<td>PIERCE</td>
			<td>#1856136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>Clonex Corp</td>
			<td>#CLEC810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Quick Draw blotting paper</td>
			<td>Sigma</td>
			<td>#P7796</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C2 platform rocking shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>8. Sonication Equipment and Reagents</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson Ultrasonic</td>
			<td>#23395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>#SOD001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche</td>
			<td>#800-363-5887</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 mM TCEP-HCl</td>
			<td>Thermo Scientific</td>
			<td>#20490</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>9. Affinity Purification Reagents and Equipment</td>
		</tr>
		<tr>
			<td>0.8 x 4 cm Bio-Rad polypropylene column</td>
			<td>Bio-Rad</td>
			<td>#732-6008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Novagen</td>
			<td>#70746</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-FLAG M2 agarose beads</td>
			<td>Sigma</td>
			<td>#A2220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calmodulin-sepharose beads</td>
			<td>GE Healthcare</td>
			<td>#17-0529-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEV protease</td>
			<td>Invitrogen</td>
			<td>#12575-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>#T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>#C2661</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>#E3889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LabQuake Shaker</td>
			<td>Thermolyne</td>
			<td>#59558</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>10. Silver Staining Reagents</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Bioshop</td>
			<td>#MET302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Bioshop</td>
			<td>t#ACE222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium-thiosulfate</td>
			<td>Sigma</td>
			<td>#S-7143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Fischer Scientific</td>
			<td>#S181-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Bioshop</td>
			<td>#FOR201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Bioshop</td>
			<td>#SOC512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>11. Reagents and Equipment for Protein Identification </td>
		</tr>
		<tr>
			<td>Trypsin Gold, Mass Spectrometry Grade</td>
			<td>Promega</td>
			<td># V5280</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 mM NH4HCO3</td>
			<td>Bioshop</td>
			<td>#AMC555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 mM CaCl2</td>
			<td>Bioshop</td>
			<td>#CCL302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>#A998-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma</td>
			<td>#F0507</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Sigma</td>
			<td>#95304</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>#16125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Millipore Zip-Tip</td>
			<td>Millipore</td>
			<td># ZTC18M960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>~10 cm of 3 &mu;m Luna-C18 resin</td>
			<td>Phenomenex</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Proxeon nano HPLC pump</td>
			<td>Thermo Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos mass spectrometer</td>
			<td>&nbsp;</td>
			<td>Thermo Fisher Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>12. Labware</td>
		</tr>
		<tr>
			<td>4 liter conical flasks</td>
			<td>VWR</td>
			<td>#89000-372</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml polypropylene falcon tubes</td>
			<td>Any Vendor</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml micro-centrifuge tubes</td>
			<td>Any Vendor</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>250 ml conical flaks</td>
			<td>VWR</td>
			<td>#29140-045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml sterile culture tubes</td>
			<td>Thermo Scientific</td>
			<td>#366052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>VWR</td>
			<td>#479-3221</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>-80 &deg;C freezer</td>
			<td>Fisher Scientific</td>
			<td>#13-990-14</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed vacuum system</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Buffers and Solutions
			1. 1 liter Terrific Broth (TB) media
			11 g Bio-Tryptone 
			22 g Yeast Extract 
			2% Glycerol 
			50 ml potassium salt stock solution
			2. Potassium Salt Stock Solution
			1.5 M K2HPO4 
			0.35 M KH2PO4
			3. Sonication Buffer
			20 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.2 mM EDTA 
			10% Glycerol 
			Before use add protease inhibitor (PI) and 0.1-0.5 mM TCEP
			4. AFC buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			5. TEV cleavage buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.2 mM EDTA 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			6. Calmodulin binding buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			2 mM CaCl2 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			7. Calmodulin wash buffer
			30 mM Tris-HCl pH 7.9 
			150 mM NaCl 
			2 mM CaCl2 
			0.1-0.5 mM TCEP
			8. Calmodulin elution buffer
			30 mM Tris-HCl (pH 7.9) 
			100 mM NaCl 
			10 mM EGTA 
			0.1-0.5 mM TCEP
			9. Developing solution (1L)
			37% Formaldehyde 
			30 g sodium carbonate 
			1000 ml distilled water
			10. Digestion buffer
			50 mM NH4HCO3 
			1 mM CaCl2
			11. Wetting and Equilibration solution
			70% acetonitrile (ACN) in 0.1% formic acid
			12. Washing solution
			100% H2O in 0.1% formic acid</td>
		</tr>
	</tbody>
</table>

identification of protein complexes in escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry

Como la mayoría de los procesos celulares están mediadas por las asambleas macromoleculares, la identificación sistemática de las interacciones proteína-proteína (PPI) y la identificación de la composición de la subunidad de multi-proteína complejos pueden dar una idea de la función de genes y mejorar la comprensión de los sistemas biológicos 1, 2. Interacciones físicas se pueden mapear con alta confianza vialarge escala aislamiento y caracterización de complejos de proteínas endógenas en condiciones próximas a las condiciones fisiológicas basado en la purificación por afinidad de proteínas cromosómicamente etiquetados en combinación con espectrometría de masas (APMS). Este enfoque ha sido aplicado con éxito en diversos organismos evolutivamente, incluyendo levaduras, moscas, gusanos, células de mamíferos, bacterias y 1-6. En particular, hemos generado una afinidad carboxi-terminal del péptido secuencial (SPA) Sistema de marcado doble para los complejos de purificación por afinidad de proteínas nativas de cultivos de bacterias gram-negativas Escherichia coli, utilizando genetically-tratables cepas huésped de laboratorio que están bien adaptados para todo el genoma investigaciones de la biología fundamental y los procesos conservados de procariotas 1, 2, 7. Nuestro SPA-tagging sistema es análogo al método de purificación de afinidad en tándem desarrollado originalmente para la levadura 8, 9, y consta de un péptido de unión a calmodulina (CBP), seguido por el sitio de escisión para la proteasa altamente específica tabaco etch virus (TEV) y tres copias del epítopo FLAG (FLAG 3X), permitiendo por dos rondas consecutivas de enriquecimiento de afinidad. Después de la amplificación casete, específicas de secuencia lineales productos de PCR que codifican el SPA-tag y un marcador seleccionable se integran y se expresa en marco como carboxi-terminal de fusiones en un fondo DY330 que es inducida para expresar transitoriamente un muy eficiente sistema de recombinación heteróloga bacteriófago lambda 10. Posterior de doble etapa de purificación usando calmodulina y anti-FLAG perlas de afinidad permite que el altamente selectivoy la recuperación eficaz de incluso complejos de baja abundancia de proteínas de cultivos a gran escala. La espectrometría de masas se utiliza para identificar los purificadores de manera estable co-proteínas con alta sensibilidad (bajos límites de detección de nanogramos). A continuación, describimos detalladas paso a paso los procedimientos que comúnmente se utilizan para el etiquetado sistemático de proteínas, purificación y espectrometría de masas basado en el análisis de complejos de proteínas solubles de E. coli, que pueden ser ampliadas y potencialmente adaptado a otras especies bacterianas, incluyendo ciertos patógenos oportunistas que son susceptibles de recombinería. Las interacciones físicas resultantes a menudo pueden revelar interesantes componentes y conexiones inesperadas que sugieren nuevos enlaces mecánicos. La integración de los datos de PPI con alternativas datos de la asociación molecular, tales como genéticos (gen-gen) las interacciones y el contexto genómico-(GC) predicciones pueden facilitar la elucidación de la organización mundial molecular de los complejos multi-proteína dentro de bivías metodológicas. Las redes generadas por E. coli se puede utilizar para obtener una perspectiva de la arquitectura funcional de los productos de genes ortólogos en otros microbios para que las anotaciones funcionales están faltando.

Once tagged bait proteins, which are expressed at endogenous levels are affinity-purified from logarithmic phase cultures the samples were run on a silver-stain gel to visualize the individual polypeptide components of the isolated stable complexes. We also subjected a second portion of the affinity-purified protein samples to gel-free tandem mass spectrometry (LCMS) to identify the corresponding polypeptide sequences. The effectiveness of this APMS procedure is shown with a representative SDS-PAGE analysis of the compon...

Watch this Scientific Journal Video about Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry at JoVE.co

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

La purificación por afinidad de las proteínas etiquetadas en combinación con espectrometría de masas (APMS) es un potente método para la asignación sistemática de las redes de interacción de proteínas y para la investigación de la base mecánica de los procesos biológicos. A continuación, se describe un péptido secuencial optimizado afinidad (SPA) APMS procedimiento desarrollado por la bacteria<em&gt; Escherichia coli</em&gt; Que se puede utilizar para aislar y caracterizar proteínas estables de múltiples complejos a cerca de homogeneidad incluso partiendo de bajo número de copias por célula.

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 ...

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Research

JoVE Journal

Biology

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

48.2K vistas.  University of Toronto. La purificación por afinidad de las proteínas etiquetadas en combinación con espectrometría de masas (APMS) es un potente método para la asignación sistemática de las redes de interacción de proteínas y para la investigación de la base mecánica de los procesos biológicos. A continuación, se describe un péptido secuencial optimizado afinidad (SPA) APMS procedimiento desarrollado por la bacteria Escherichia coli Que se puede utilizar para aislar...

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

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

El análisis de grandes complejos de proteínas por espectrometría de masas estructurales

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

La cuantificación de proteínas mediante el enriquecimiento de inmunoafinidad péptido acoplado a espectrometría de masas

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

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 ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

El Protocolo para la identificación de las interacciones proteína-proteína base de<sup&gt; 15</sup&gt; N metabólico Etiquetado, inmunoprecipitación, cuantitativa Espectrometría de Masas y Modulación Affinity

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

La identificación de la interacción proteína-proteína en<em&gt; Drosophila</em&gt; Jefes adultas por Tandem Affinity purificación (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

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 ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Las multifacéticas beneficios de la proteína co-expresión en<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...