SW y TMV son apoyados por la CNRR y el NHLBI en los Institutos Nacionales de Salud. BSK SA y son apoyados por el NINDS y NIGMS de los Institutos Nacionales de Salud.

PROCEDIMIENTO EXPERIMENTAL El procedimiento experimental (Fig. 2) consta de cuatro partes que se describen de una manera gradual a continuación. Parte 1: Subclonación y la expresión de la C-terminal de los receptores P2X2. Hemos expresado la longitud C-terminal de los receptores P2X2 en las bacterias para identificar las proteínas del cerebro al que se une. <ol><li> El C-terminal (residuos 353-472) de los receptores P2X2 (Fig. 1) fue amplificado por PCR, clonado en pGEX 4NT1 (GE Life Sciences) y se verificó con la secuenciación. </li><li> El plásmido recombinante se transformó en E. Coli (BL21) para la expresión de proteínas recombinantes. </li><li> Stocks de glicerol de bacterias (E. coli BL21) que contiene el P2X2 CT-GST plásmido se raspa con una punta de pipeta estéril y se inoculó en 5 ml de medio Luria-Bertani (LB) los medios de comunicación con el marcador adecuado selectiva (50 g / ml ampicilina) y incubaron durante la noche en un agitador orbital a 37 º C. </li><li> Las culturas crecido durante la noche se inocularon en 250 ml de medio LB con los antibióticos adecuados (50 mg / ml ampicilina) y se incubaron en un agitador orbital a 250 rpm durante 2-3 horas a 37 º C hasta que la densidad óptica a 600 nm alcanzó ~ 0.6-0.7. </li><li> La cultura fue inducida con 1 mM de IPTG y se incubaron a 37 º C durante 3 horas para la expresión de la proteína. </li><li> La cultura se separó luego a 5000 g durante 15 min a 4 ° C (1 ml de la cultura se ha guardado para analizar el nivel de expresión y etiquetados como &quot;inducida&quot;). Sobrenadante fue descartado y el pellet se resuspendió en 20 ml de solución salina de Tris-EDTA (STE) buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA pH 8,0). </li><li> La suspensión se centrifugó a 5000 g durante 15 minutos en un tubo Falcon de 50 ml. El sobrenadante fue descartado y el pellet semi-seco se congeló a-70C durante la noche. </li><li> El-70C se resuspendió en 40 ml de solución amortiguadora de lisis fría (2% (w / v) sarkosyl, 15 mg de lisozima, 150 mM NaCl, 50 mM Tris pH 7,5, 5 mM de TDT y la tableta completa inhibidor de la proteasa) , y la suspensión se incubó en hielo durante 30 min. Durante la incubación, 3 ml de glutatión-Sepharosa 4B cuentas se lavaron con 20 ml de tampón fosfato salino (PBS) y 20 ml T-PBS (0,1% (v / v) TritonX-100 en PBS), seguido por PBS. Las cuentas se resuspendieron en 1 ml de PBS. </li><li> La suspensión bacteriana se congelarse y descongelarse 3 veces en nitrógeno líquido y se sonica (1s de encendido / apagado de 30 micrones de tres minutos sobre el hielo) y se incubaron durante 30 minutos con un 4% (v / v) de Triton X-100, 10 mM MgSO 4 y 2 mM ATP en el hielo. </li><li> El lisado se centrifugó a 20.000 g durante 15 min a 4 ° C y el precipitado se descartó (una muestra de pellet y 1 ml de sobrenadante se guardó para SDS-PAGE para comprobar las cantidades de proteínas en el citosol y las membranas). El sobrenadante se incubó con las cuentas de una hora (o toda la noche) a 4 ° C. </li><li> Las bolas se giró a 500 g por 5 min y se descartó el sobrenadante (muestra se salvó de fracción libre). Las bolas se lavaron con PBS-T en cinco ocasiones (lavados se salvaron de los controles de lavado). </li><li> Las cuentas se resuspendieron en PBS a dar el 50% (w / v) los purines y se almacena en el refrigerador a 4 º C hasta su uso. La concentración de proteínas se estimó por el ensayo de Bradford (siguiendo las instrucciones del fabricante). </li></ol> Parte 2: Preparación de los lisados ​​de todo el cerebro P2X2 receptores son conocidos por ser altamente expresado en el cerebro 8. En el presente análisis, hemos tratado de identificar las proteínas que interactúan con los receptores P2X2 a partir de lisados ​​de cerebro de rata entera (Fig. 3). <ol><li> Cerebro de rata adulta fue cosechada (animales fueron sacrificados de acuerdo con un IAACUC UCLA-protocolo aprobado y de acuerdo con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio) y aclararán inmediatamente helada PBS (3 veces). </li><li> Para lisar las células de 10 ml (~ 5 veces el peso húmedo del cerebro cosecha) de tampón de lisis helada que contiene 150 mM NaCl, 50 mM Tris-HCl, pH 7,4, 10 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na 3 VO 4, 1 mM PMSF, 5 mg / ml de leupeptina, 1% (v / v) NP-40, y una tableta de inhibidor de la proteasa cóctel fue introducido en el cerebro cosechado. </li><li> El tejido cerebral se homogeneizó en el hielo con un homogeneizador de vidrio Dounce hasta obtener una mezcla de consistencia homogénea se logró. </li><li> Lisado celular se centrifugó a 2000 g durante 5 minutos para eliminar las células sin romper. El sobrenadante se recogió y se volvió de nuevo a 29.000 g durante 60 minutos para eliminar orgánulos intactos y agregados de la membrana. </li><li> Inmediatamente después de la centrifugación, el sobrenadante se transfirió a un tubo limpio. La concentración de proteínas del lisado de todo el cerebro se midió por el ensayo de Bradford (normalmente ~ 15 mg / ml). </li><li> El lisado se dividió en alícuotas y se almacenaron a-70C. </li></ol> Parte 3: Aislamiento de P2X2 C-cola proteínas asociadas Para identificar a los socios de unión de P2X2 CT-GST a partir de lisados ​​de todo el cerebro, un desplegable ensayo se realizó mediante el uso de CT-GST inmovilizado en glutatión sefarosa 4B cuentas como cebo. Para los controles, solo GST unida a las microesferas se utilizó. <ol><li> Cerebro lisado se precleared con cuentas de IVA durante 1 hora a 4C. El sedimento se desechó y el sobrenadante se utilizó para otros análisis. </li><li> Igual cantidad de GST y P2X2 CT-GST (10 mg) con destino a glutatión sefarosa 4 cuentas B se incubaron durante la noche con 100 g de proteínas precleared solubilizados lisado de cerebro de rata entera, con la rotación a 4 º C en el tampón de lisis. </li><li> Las cuentas se hace girar a 1000 g por 5 min y el sobrenadante fue descartado. </li><li> Las bolas se lavaron una vez con el tampón de lisis y se hierve en modificar Laemli buffer [4% (w / v) SDS, 10% (v / v) 2-mercaptoetanol, 2% (v / v) de glicerol, 0,004% (w / v) azul de bromofenol y 0,125 M pH 6,8 TrisCl] durante 5 min. </li><li> Las muestras se centrifugaron a 5000 g por 5 min y el sobrenadante se separó en un 10% SDS-PAGE (Fig. 4). Los geles fueron teñidos con gel de proteínas SYPRO Rubí mancha (de acuerdo con las instrucciones del fabricante) y se visualizaron bajo luz ultravioleta. P2X2 CT proteínas asociadas fueron comparados con los recuperados utilizando GST de cero como cebo. Cuatro réplicas biológicas fueron realizadas (es decir, cuatro cerebros y cuatro bajas tirar independiente). </li></ol> Parte 4: Identificación de proteínas. Proteínas separadas por electroforesis en gel fueron extirpados del gel y se identifican por espectrometría de masas 12. Nota: la ausencia de polvo durante todo el proceso es fundamental para reducir la contaminación de la queratina. El experimentador debe usar una mascarilla, red para el cabello y guantes en todo momento y nunca toque la región de gel de interés sin el uso de guantes. <ol><li> Todas las bandas de proteínas visible recuperado de la fuerza por P2X2 fueron extirpados con una cuchilla limpia y cortada en cubitos (Fig. 4). Regiones correspondientes del gel en la GST-pull nula por el carril también fueron extirpados de la misma manera sin tener en cuenta a las manchas de banda (bandas específicas para el cebo de GST-nulos fueron extirpados también e identificados), para asegurar que la presencia de proteínas por debajo del nivel de sensibilidad de la tinción no se ha perdido. </li><li> Las piezas de gel se incubaron con 200 l de bicarbonato amónico 50 mM en el 50% (v / v) acetonitrilo (ACN) y los tubos vortex durante 15 min a temperatura ambiente. Repita este paso de lavado. </li><li> Las piezas de gel se deshidrata mediante la adición de 200 l de acetonitrilo (piezas de gel se encogen y se vuelven opacas en color). </li><li> Acetontrile fue removido y las piezas de gel se seca en un speedvac de ~ 10 min. </li><li> Las piezas de gel fueron incubadas con 30 l de la recién preparada 10 mM Tris DTT/10 mM (2-carboxi-etil) clorhidrato de fosfina (TCEP) de un volumen suficiente para sumergir las piezas. Esto se dejó durante 30 minutos a baño de agua 56C. </li><li> A continuación la solución de DTT fue reemplazado con 100 l de bicarbonato de amonio 500 mM en una solución de acetonitrilo al 50% y las rodajas de gel se lavó con bicarbonato ammounium. </li><li> La solución de lavado fue aspirado y 200 l de acetonitrilo añadido a la deshidratación de los geles. </li><li> Acetonitrilo fue eliminado y 100 l recién preparada una solución de 100 mM yodoacetamida se añadió para alquilar sulfhidrilos libres. Este paso procedió durante 25 minutos a temperatura ambiente en la oscuridad. </li><li> Solución de yodoacetamida fue removido y las piezas de gel se lavaron con 200 l de bicarbonato amónico 50 mM en el 50% acetontrile (pH 8,0) durante 2 minutos, mientras agitación. Esta etapa de lavado se repitió. </li><li> Después de retirar la solución de lavado, las piezas de gel se incubaron con 200 l de acetonitrilo que se eliminó por speedvac de ~ 10 minutos. </li><li> En este paso las piezas de gel está listo para la digestión de tripsina. Piezas de gel se incubaron en 30 l de solución de tripsina (20 ng / l de concentración de trabajo) y se coloca en hielo durante 10 minutos para los geles de estar bien hinchado. Tripsina en exceso se eliminó y se rehidrata partículas de gel recubierto con 30 ml con bicarbonato amónico 50 mM para mantenerlos sumergidos a lo largo de la digestión que se le permitió continuar por 12 a 16 horas a 37 º C. </li><li> L Cinco de 5% (v / v) de ácido fórmico se añade a desactivar la digestión de la tripsina y la detención. </li><li> Los tubos se agitaron durante aproximadamente 15 min y se centrifuga brevemente para llevar el líquido a la parte inferior del tubo, que fue trasladado a un ml frasco limpio y etiquetado 0.5. </li><li> Próximos 30 l de 0,1% (v / v) de ácido fórmico en el 50% de acetonitrilo se añadió a piezas de gel por lo que fueron cubiertos solo dos veces seguido por agitación durante 10-15 minutos, separados por centrifugación breve. Este paso se repitió una vez, en sustitución del ácido fórmico y acetonitrilo entre los pasos. </li><li> El líquido final fue eliminado y se combinan todas las solución de extracción en un solo vial. Este volumen se redujo a aproximadamente 30 uL en el speedvac. La muestra estaba listo para la fase inversa nano-cromatografía líquida y la identificación de proteínas por espectrometría de masas. En este ejemplo concreto, este paso se lleva a cabo en un espectrómetro de masas en tándem Orbi-trampa de ThermoFclasificador (elegido por su alta precisión la masa, el ciclo de trabajo rápido para la detección de las proteínas y las características de funcionamiento en general robusta), aunque otros modelos e instrumentos de los fabricantes podría ser, junto con el enfoque descrito en este punto. </li><li> Los detalles y los criterios de análisis de espectrometría de masas son ahora una breve presentación. Todos los péptidos fueron separados de fase inversa nano-LC (Eksigent) y los péptidos fueron cargados en un C18 columna de fase inversa, con un caudal de 3 l / min. Una fase móvil fue de 0,1% de ácido fórmico y 2% de ACN en el agua; B de la fase móvil fue de 0,1% de ácido fórmico y el 20% de agua en ACN. Los péptidos se eluyeron de la columna a un flujo de 220 nl / min con un gradiente lineal de B un 5% a 50% de B durante 90 minutos, y luego a 95% de B durante 5 min y, finalmente, manteniendo constante B del 95% durante 5 minutos . Los espectros fueron adquiridos en los datos de modo dependiente de la Orbi-trampa que se utiliza para los análisis de MS y LTQ de MS / MS análisis. Los péptidos fueron identificados por búsquedas en los espectros de la base de datos de ratas IPI v.3.53 utilizando el algoritmo SEQUEST integrado en el paquete de software BioWorks. Cada péptido se reunieron los siguientes criterios: XCorr ≥ 2 (+1), ≥ 3 (2), ≥ 4 (3), y DeltaCN&gt; 0.1. Todos los espectros utilizados para la identificación de proteínas fueron inspeccionados de forma manual y no las proteínas aceptada cuando menos de dos péptidos fueron identificados. Sólo las proteínas presentes tras cada uno de los cuatro P2X2 bajas tirar y tirar ausente durante bajadas GST-fueron considerados nulos para otros análisis. </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="la figura 1" /> Figura 1. Representación esquemática de una subunidad del receptor P2X2. A. La caricatura muestra la topología de una subunidad del receptor P2X2. El dominio citosólico se compone de N y C terminales. El C-terminal de P2X2 receptor (rojo) se utiliza como cebo para tirar hacia abajo del ensayo. B. secuencia de aminoácidos del receptor P2X2 C-terminal utilizado en este estudio. <img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> Figura 2. Diagrama de flujo y la línea de tiempo del protocolo usado para la expresión, purificación, tira hacia abajo y la identificación de las proteínas. Mostramos el esquema del protocolo con la línea de tiempo. Rata adulta lisado cerebro estaba preparado fresco inmediatamente antes del uso de la fuerza hacia abajo ensayos. <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> Figura 3. Representación esquemática de los análisis proteómicos de binarios P2X2 C-terminal de la red en el cerebro de la rata. El C-terminal de los receptores P2X2 fusionada con la proteína GST unida a las microesferas GST fue utilizado para tirar hacia abajo los ensayos. Cerebro lisado fue preparado y las proteínas se incubaron con las proteínas recombinantes inmovilizado. Fracción libre se lavó con el tampón de lisis. Se identificaron las proteínas por espectrometría de masas. <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> Figura 4. Identificación de los socios de la unión del C-terminal de los receptores P2X2. Gel Sypro manchado muestra un espectro de proteínas putativo que interactúan con el C-terminal de los receptores de fusión con GST (caja azul). Carriles de los controles de GST sola (recuadro amarillo) y cuentas de glutatión sefarosa solo se muestran también. Las flechas indican ejemplos de bandas únicas que fueron extirpados para su posterior análisis por espectrometría de masas.

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Los canales iónicos son una clase importante de proteínas integrales de membrana. Que contienen poros llenos de agua que permiten selectivamente el movimiento de iones por sus gradientes electroquímicos a través de la membrana plasmática. Los canales iónicos puerta entre abierta y cerrada de los estados. El paso de disparo es activado por los transmisores (por ejemplo, ATP) en el caso de los canales iónicos P2X ligando, o puede ser regulada por las interacciones con otras proteínas. La última década ha sido te...

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<tr>
<td>Acetonitrile</td>
<td>Reagent</td>
<td>JT Baker</td>
<td>9829-02</td>
<td>&nbsp;</td>
<tr>
<td>Acrylamide</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0156</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1507-01</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
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<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
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<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
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<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
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<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
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<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
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<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
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<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

Ligando la base de los canales iónicos de comunicación sináptica en el sistema nervioso 1. En los mamíferos hay tres familias de ligandos de canales: el lazo cys, el glutamato y los canales de los receptores P2X 2. En cada caso, la unión de emisor lleva a la apertura de un poro por donde fluyen los iones de sus gradientes electroquímicos. Muchos ligando los canales también son permeables a los iones de calcio 3, 4, que tienen funciones de señalización hacia abajo 5 (por ejemplo, la regulación de genes) que pueden superar la duración de la apertura del canal. Así, ligando los canales pueden ser señal en escalas de tiempo amplio que van desde unos pocos milisegundos a días. Teniendo en cuenta estas importantes funciones, es necesario entender cómo ligando los canales iónicos se están reguladas por las proteínas, y cómo estas proteínas pueden sintonizar señales. Estudios recientes sugieren que muchos, si no todos, los canales pueden ser parte de complejos de proteínas de señalización 6. En este artículo se explica cómo identificar las proteínas que se unen a los aspectos C-terminal del dominio del receptor P2X2 citosólica. Receptores P2X ATP-dependientes canales de cationes y constará de siete subunidades (P2X1-P2X7). Receptores P2X se expresan ampliamente en el cerebro, donde median la transmisión sináptica excitatoria y la facilitación presináptica de la liberación de neurotransmisores 7. Receptores P2X se encuentran en las células excitables y no excitables y mediar un papel clave en la señalización neuronal, la inflamación y la función cardiovascular 8. P2X2 receptores son abundantes en el sistema nervioso 9 y son el foco de este estudio. Cada subunidad P2X se cree que tiene dos segmentos que atraviesan la membrana (TM1 y TM2), separadas por una región extracelular intracelular 7 y N y C terminales (Fig. 1a) 7. Subunidades P2X 10 (P2X1-P2X7) muestran un 30-50% de homología de secuencia en el nivel de aminoácidos 11. Receptores P2X contienen sólo tres subunidades, que es el más simple estequiometría entre los receptores ionotrópicos. El P2X2 C-terminal consta de 120 aminoácidos (Figura 1b) y contiene varias proteínas consenso sitios de atraque, que apoya la hipótesis de que P2X2 receptor puede ser parte de complejos de señalización. Sin embargo, a pesar de varias funciones han sido atribuidas a la C-terminal de los receptores P2X2 nueve ningún estudio ha descrito los socios molecular que se acoplan a la parte intracelular de esta proteína a través de toda la longitud C-terminal. En este artículo se describen los métodos de una aproximación proteómica para identificar las proteínas que interactúan con el cuerpo entero C-terminal de los receptores P2X2.

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Se describe un protocolo simple de identificar a las proteínas del cerebro que se unen a la terminal C de longitud completa de la ATP-dependientes P2X2 receptores. La extensión y aplicación sistemática de este enfoque a todos los receptores P2X se espera que conduzca a una mejor comprensión de la señalización del receptor P2X.

Proteómica para identificar proteínas que interactúan con los canales de cationes P2X2 ligando

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K vistas.  David Geffen School of Medicine, University of California, Los Angeles. Se describe un protocolo simple de identificar a las proteínas del cerebro que se unen a la terminal C de longitud completa de la ATP-dependientes P2X2 receptores. La extensión y aplicación sistemática de este enfoque a todos los receptores P2X se espera que conduzca a una mejor comprensión de la señalización del receptor P2X.

Video: Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

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

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - un método para identificar transcriptoma en todo los sitios de unión de las proteínas de unión a ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Usando alcance para identificar posibles motivos de reglamentación en los genes coregulated

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

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 ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado levadura sistema híbrido de dos para identificar proteínas que interactúan con el factor de crecimiento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

Invierta levadura de dos sistema híbrido para identificar mamíferos Receptor Nuclear Residuos que interactúan con ligandos y / o antagonistas

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

De abajo hacia arriba y de escopeta Proteómica para identificar una exhaustiva Coclear proteoma

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Captura Glicopéptido Proteómica Superficie Celular

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.