We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1. Preparación de lisado <ol><li> Crecer P. aeruginosa células en LB a la DO deseada. NOTA: Para obtener orientación: utilizar ≈ 100 ml de cultivo / muestra para cultivos en fase estacionaria y ≈ 500 ml cultur / muestra para cultivos en fase log (OD 600 nm = 0,5). </li><li> Pellet por centrifugación durante 20 min a 5000 x g. </li><li> Resuspender 0,5-1 g de sedimento en 1 ml de tampón de lisis (MES 6,7 mM, 6,7 mM de HEPES, NaCl 200 mM, 6,7 mM Kac, DDT 1 mM, pH 7,5) y se añade inhibidor de la proteasa (mini completo, libre de EDTA), así como DNaseI. </li><li> Lisan las células mediante 3 pasos a través de una célula de presión francesa, a 20.000 psi (ver Lista de Materiales). </li><li> Ultra-centrifugar el lisado celular a 100.000 x g durante 1 hora a 4 ° C. </li><li> Guardar el sobrenadante (vaya al paso 2). </li><li> Lavar el sedimento con 1 ml de 1x tampón de lisis pipeteando arriba y abajo. </li><li> Ultracentrífuga a 100.000 x g durante 1 hora a 4 ° C. </li><li> Flash congelar el sedimento ennitrógeno líquido y se almacena a -20 ° C hasta su utilización para la captura de las proteínas de membrana (ver paso 3). </li></ol> 2. La eliminación de los nucleótidos libres c-di-GMP y Otros (Fracción Soluble solamente) <ol><li> Lavar una columna de desalación PD10 (ver Lista de Materiales) con 10 ml de tampón de lisis frío. </li><li> Verter el sobrenadante (≈ 1 ml) en el PD10 con el fin de eliminar los nucleótidos. </li><li> Se eluye con 4 ml de tampón de lisis frío (500 l pasos). </li><li> Seleccione las fracciones más concentradas como se determina mediante el ensayo de Bradford, y los mismos se agrupen. </li></ol> 3. Pellet resuspensión y solubilización (fracción de membrana solamente) <ol><li> Resuspender el precipitado en 500 a 1.000 l de búfer de captura 1x (sin detergente) (ver Tabla 1). NOTA: El pellet es difícil para volver a suspender. Se aconseja primera pipeta de arriba abajo con una pipeta para volver a suspender el sedimento o menos, luego de usar una jeringa de 27 G para así homogeneizar la solución. </li><li> Añadir 1% (w / v) n-dodecil-β-D-maltopiranósido (DDM). </li><li> Incubar a 4 ° C durante al menos 2 horas (o O / N) en una rueda giratoria. </li><li> Ultracentrífuga a 100.000 x g durante 1 hora a 4 ° C. </li><li> Se recoge el sobrenadante. </li></ol> 4. Proteína de medición de concentración <ol><li> Medir la concentración de proteína por el ensayo de Bradford (mediante ensayo BCA para la fracción de membrana). </li><li> Ajuste la concentración de proteína total a 10 mg / ml. </li></ol> 5. Captura <ol><li> Mezclar 300 g de proteína con 1 mM del PIB, GTP, ATP, CTP, y 20 l de tampón de captura de 5x (mM HEPES 100 mM, KAc 250 mM, MgAc 50, 5% de glicerol, pH 7,5) (Tabla 1). Ajuste el volumen de reacción de 100 l con H 2 O. NOTA: el volumen total incluye el volumen del CDG-CC y c-di-GMP en el caso del control de la competencia (véase el paso 5.3 y la Tabla 2). NOTA: todos los experimentos se llevaron a cabo en 200 y# 181; l tiras de PCR de 12 tubos (Thermo Scientific). NOTA: para la fracción de membrana, asegúrese de mantener siempre la concentración DDM encima de la concentración micelar crítica (0,01% w / v) hasta la etapa de solubilización de proteínas en urea 8 M (Paso 7.1). </li><li> Incubar a 4 ° C durante 30 min en una rueda giratoria. </li><li> Añadir 10 mM CDG-CC (concentración final). NOTA: incluir un control sin CDG-CC (referido como &quot;el control del grano&quot;), y un control suplementado con 1 mM de c-di-GMP (&quot;control de la competencia&quot;) (ver Tabla 2). NOTA: la concentración CDG-CC se puede ajustar de 1 a 10 mM. </li><li> Incubar a 4 ° C durante al menos 2 h (O / N para la fracción de membrana) en una rueda giratoria, en la oscuridad. </li><li> Después de un giro corto, cross-link mediante la activación del resto reactivo de la CDG-CC con luz UV durante 4 min, utilizando un CaproBox (ver Lista de Materiales, λ = 310 nm, irradiancia ≥10 mW / cm², distancia de la fuente = 2 cm). Nota: Retire la tapa de las tiras de reticulación previa. </li><li> Añadir 25 l de 5x tampón de lavado (NaCl 5 M, Tris 250 mM, pH 7,5) y 50 l de perlas magnéticas de estreptavidina así resuspendidos, homogeneizar suavemente. </li><li> Incubar a 4 ° C durante 1 hora en una rueda giratoria. </li></ol> 6. Los pasos de lavado NOTA: (Imán: ver Lista de Materiales). Comience con una captura de las perlas magnéticas en la tira tapa PCR, con el imán. A continuación, reemplace la tira de PCR por uno nuevo que contenga la siguiente solución de lavado. Retire el imán y volver a suspender las perlas, y se incuba 2 min. Centrifugar y reemplazar la tapa por una tapa fresco. <ol><li> Las etapas de lavado (fracción soluble únicamente) <ol><li> Lavar 6 veces en 200 l de tampón de lavado 1x. </li><li> Lavar una vez en 200 l de calidad HPLC H 2 O. </li><li> Lavar 6 veces en 200 l 80% de acetonitrilo. </li><li> Lavar 2 veces en 200 l de calidad HPLC H 2 O. </li></ol></li><li> Las etapas de lavado (membrafracción ne solamente) <ol><li> Lavar 5 veces en 200 l de tampón de lavado 1x + 0,1% DDM. </li><li> Lavar 2 veces en 200 l de tampón de lavado 1x + 0,05% DDM. </li><li> Lavar una vez en 200 l de tampón de lavado 1x + 0,025% DDM. </li><li> Lavar una vez en 200 l de tampón de lavado 1x + 0,0125% DDM. </li><li> Lavar 3 veces en 200 l mM ABC 100 (bicarbonato de amonio, NH 4 CO 3) + 2 M urea. </li></ol></li></ol> 7. MS Preparación de la muestra <ol><li> Resuspender las perlas (directamente en la tapa) en 20 l mM ABC 100 (mM ABC 100 + 8 M urea para la fracción de membrana) y transferir a tubos de 1,5 ml. </li><li> Fracción de membrana única: se incuba a 60 ° C durante 5 min, agitando a 500 rpm. </li><li> Añadir 0,5 l mM TCEP 200 (tris (2-carboxietil) fosfina) y se incuba a 60 ° C durante 1 hora, con agitación a 500 rpm. Enfriar a 25 ° C. </li><li> Añadir 0,5 l de 400 mM recién preparada yodoacetamida y se incuba a 25 ° C durante 30 minutos, agitando unaT 500 rpm y en la oscuridad. </li><li> Añadir 0,5 l 0,5 M N-acetil-cisteína y se incuba a 25 ° C durante 10 min, agitando a 500 rpm. </li><li> Fracción de membrana única: añadir 1 l Lys-C e incubar a 37 ° C, O / N. </li><li> Añadir 2 g de tripsina y se incuba O / N a 37 ° C, agitando a 500 rpm (wrap en Parafilm para evitar el secado). NOTA: las muestras se pueden almacenar a -20 ° C en esta etapa. NOTA: fracción de membrana solamente: añadir mM ABC 100 para ajustar la concentración de urea a &lt;2 M antes de la adición de tripsina. </li><li> Brevemente centrifugar los tubos y recoger bolitas con el imán. </li><li> Transferir el sobrenadante a un nuevo tubo de 1,5 ml (repita este paso si los granos se dejan). </li><li> Añadir 5 l 5% de TFA (ácido trifluoroacético) + 1 l 2 M HCl (15 l 5% TFA + 5 l 2 M HCl para la fracción de membrana). </li><li> Condición columnas C18 MicroSpin (El Grupo Nest, MA, EE.UU.) con 150 l de acetonitrilo (spin 20 segundos a 2.400 rpm). </li><li> Equilibrate las columnas C18 2 veces con 150 l 0,1% TFA (giro 20 s, 2400 rpm). </li><li> Cargue la muestra y girar 2 min, 2000 rpm. </li><li> Actualizar el flujo a través en la columna y repetir la etapa de hilatura (2 min, 2000 rpm). </li><li> Lavar 3 veces con 150 l 0,1% de TFA, 5% de acetonitrilo (20 segundos, 2400 rpm). </li><li> Tomar un tubo nuevo y se eluye dos veces con 150 l 0,1% de TFA, 50% de acetonitrilo (2 min, 2000 rpm). </li><li> Seque los péptidos en una velocidad-vac. </li><li> Resuspender en 40 l 98% H 2 O, 2% de acetonitrilo, ácido fórmico 0,15%. </li><li> Sonicar 20 seg (ciclo de pulso 0,5, amplitud 100%; ver Lista de Materiales) y centrifugar 5 seg, 12.000 rpm (centrífuga de mesa). Vortex 10 seg, y de girar 5 seg, 12.000 rpm. Transferencia en un vial de HPLC para el análisis de LC-MS / MS. </li><li> Congelar a -20 ° C. NOTA: las muestras se pueden almacenar a -20 ° C en esta etapa. </li></ol> 8. LC-MS / MS Analysis <ol><li> Ejecutar un nano-LC (sistemas nano-LC) equipped con una columna RP-HPLC (75 m x 37 cm) rellena con resina C18 (magia C18 AQ 3 m) usando un gradiente lineal de disolvente A (ácido fórmico al 0,15%, 2% de acetonitrilo) 95% y 5% de disolvente B ( 98% de acetonitrilo, ácido fórmico 0,15%) a 35% de disolvente B durante 60 min a un caudal de 0,2 l / min. </li><li> Analizar los péptidos utilizando LC-MS / MS (doble presión LTQ-Orbitrap Velos espectrómetro de masas, conectado a una fuente de iones de electrospray). NOTA: El modo de adquisición de datos se creó para obtener una alta resolución MS Analizar en la parte FT del espectrómetro de masas con una resolución de 60.000 FWHM seguido por MS / MS de las exploraciones en la trampa de iones lineal de los 20 iones más intensos. Para aumentar la eficiencia de los intentos de MS / MS, el modus proyección estatal encargada estaba habilitado para excluir sin asignar y solitariamente cobra iones. Colusión disociación inducida se desencadenó cuando el precursor superó los 100 recuentos de iones. La duración dinámica de exclusión se fijó a 30 seg. El tiempo de acumulación de iones se fijó a 300 mseg (MS) y 50mseg (MS / MS). </li></ol> 9. Base de datos de búsqueda <ol><li> Descargue el P. aeruginosa NCBI-base de datos a través de la página de NCBI ( <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ). </li><li> Convertir los espectros MS prima en archivos genéricos de la mascota (MGF) utilizando la herramienta de conversión MassMatrix ( <a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.py</a> ). </li><li> Buscar en este archivo mgf usando MASCOTA versión 2.3 contra la P. aeruginosa NCBI-base de datos que contiene adelante y entradas de proteínas inversa señuelo. </li><li> Realizar una en la digestión de tripsina silico después de lisina y arginina (a no ser seguido por prolina) tolerando dos perdidas divisiones en péptidos plenamente tríptico. </li><li> Establezca los parámetros de búsqueda de base de datos para permitir metioninas oxidado (15.99491 Da) como variable modificaciones y carboxiamidometilación (57.021464 Da) de residuos de cisteína como fijo modificación. Para MASCOTA nos buscaing escaneos de alta resolución, establecer la tolerancia de masa precursor de 15 ppm y establecer la tolerancia de masa fragmento de 0,6 Da. Por último, establezca la proteína FDR al 1%. </li><li> Importe las búsquedas de la mascota de P. experimentos aeruginosa CCMS en Andamios (Proteomesoftware, Versión 3), establecen los parámetros para obtener una proteína FDR cercano al 1%, y extraer los recuentos espectrales totales. </li><li> Para que los resultados representativos presentan en este documento, se utilizó una prueba t pareada para comparar el experimento con el control de la competencia, y sólo se consideran golpes con un valor de p inferior a 0,1, y un índice de recuento espectral por encima del 2 (experimentar recuentos espectrales / espectral control de la competencia recuentos). </li><li> Los datos se pueden exportar en cualquier software de hoja de cálculo para su posterior análisis. </li></ol> 10. Cuantificación-Label gratis <ol><li> Importe los archivos RAW en el software progénesis LC-MS (Dinámica no lineal, versión 4.0). </li><li> Realizar LC-MS alineación y detección de características en setti defectoNGS. </li><li> Exportación de los datos en formato mgf de progénesis LC-MS. </li><li> Buscar en los espectros MS / MS utilizando la mascota contra el NCBI P. base de datos que contiene aeruginosa hacia adelante y entradas de proteínas inversa señuelo. </li><li> Importe los resultados de búsqueda de base de datos en progénesis LC-MS y un mapa de los péptidos identificaciones a características MS1. </li><li> Para la evaluación de datos (cálculo de los niveles de significación, plegables cambio proporciones) se utilizó el guión ProteinSQAnalysis de SafeQuant (umbral: el valor q por debajo de 0,1, índice de recuento espectral superior a 2). </li></ol>

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The authors have nothing to disclose.

Especial cuidado se debe tomar en varias etapas del protocolo. La concentración de proteínas es un parámetro crítico con una concentración de 10 mg / ml siendo difícil de alcanzar cuando las células se cultivan bajo condiciones de crecimiento específicas (por ejemplo biofilms o pequeñas variantes de colonias). Por lo tanto, la resuspensión de pellets se debe realizar en un bajo volumen de tampón de lisis. Las concentraciones de proteína se pueden reducir a 8 mg / ml. En comparación con el método p...

c-di-GMP es un segundo mensajero clave usada por la mayoría de las bacterias para controlar varios aspectos de su crecimiento y comportamiento. Por ejemplo, c-di-GMP regula la progresión del ciclo celular, la motilidad y la expresión de exopolisacáridos y adhesinas de superficie 2-4. A través de la coordinación de tales procesos de c-di-GMP promueve la formación de biofilm, un proceso que está asociado con infecciones crónicas de una gama de bacterias patógenas 5. c-di-GMP se sintetiza por enzimas llamadas guanilato diguanylate (PED) que albergan un dominio catalítico GGDEF 4. Algunos PED poseen un sitio de inhibidor que regula hacia abajo la actividad de la adenilato sobre c-di-GMP de unión. La degradación de c-di-GMP está catalizada por dos clases distintas de las fosfodiesterasas (PDE) que alberga ya sea un EAL catalítica o HD-GYP de dominio 6,7. La mayoría de las proteínas efectoras conocidas que se unen directamente c-di-GMP pertenecen a una de las tres clases de proteínas: catalíticaGGDEF o EAL dominios aliado inactivos y dominios Pilz, pequeños interruptores moleculares que experimentan cambios conformacionales a vinculantes c-di-GMP 8. PED, PDE y Pilz proteínas están bien caracterizados y sus dominios se pueden predecir en silico relativamente segura. Un especial interés se centra ahora en la identificación de nuevos tipos de efectores c-di-GMP. Algunos efectores de c-di-GMP con diferentes motivos de unión se describieron recientemente como el CRP / FNR Bcam1349 familia de proteínas en Burkholderia cenocepacia o la FleQ regulador transcripcional en P. aeruginosa 9,10. Además, riboswitches-GMP-específico c-di han sido recientemente identificados y muestran para controlar la expresión génica de una manera c-di-GMP-11 dependiente. Los motivos c-di-GMP de unión de diferentes efectores son sólo mal conservadas hacer predicciones bioinformáticas de tales proteínas difícil. Para solucionar este problema, hemos desarrollado un método bioquímico, que se basa en el uso de un spe c-di-GMPcaptura compuesto espe- combinada con espectrometría de masas 1,12,13. Hemos diseñado recientemente una captura compuesto c-di-GMP novela trivalente (CDG-CC, la Figura 1) 1. Este andamio química se compone de: 1) un resto de c-di-GMP utilizado como cebo para capturar c-di-GMP proteínas de unión, 2) un grupo reactivo fotoactivable-UV utiliza para reticular el CDG-CC a las proteínas unidas y 3) una biotina para aislar las proteínas capturadas usando perlas magnéticas recubiertas con estreptavidina. El CDG-CC se puede utilizar para capturar directa y específicamente efectores de c-di-GMP a partir de una mezcla compleja de macromoléculas como lisados ​​celulares. Compuesto de captura basada y han sido previamente informado enfoques químicos proteómica basadas ser aplicable a una amplia gama de organismos, por ejemplo, Caulobacter crescentus, Salmonella enterica serovar typhimurium y P. aeruginosa 1,14. En este trabajo metodológico,proporcionamos una descripción en profundidad del procedimiento CCMS utilizando extractos de P. aeruginosa como un ejemplo. Este estudio establece CCMS como una herramienta poderosa y versátil para identificar bioquímicamente nuevos componentes que participan en la señalización de pequeña molécula.

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

Se ha logrado un progreso considerable en la última década hacia la identificación y caracterización de las enzimas implicadas en la síntesis (guanilato diguanylate) y degradación (fosfodiesterasas) del segundo mensajero c-di-GMP. Por el contrario, hay poca información disponible acerca de los mecanismos moleculares y componentes celulares a través del cual esta molécula de señalización regula una amplia gama de procesos celulares. La mayoría de las proteínas efectoras conocidas pertenecen a la familia Pilz o se degeneró guanilato diguanylate o fosfodiesterasas que han renunciado a la catálisis y han adoptado la función efectora. Por lo tanto, para definir mejor la red de c-di-GMP celular en una amplia gama de bacterias se requieren métodos experimentales para identificar y validar nuevos efectores para el cual fiable en las predicciones in silico fallar. Recientemente hemos desarrollado una novela de captura compuesto Espectrometría de Masas (CCMS) tecnología basada como una poderosa herramienta parabioquímicamente identificar y caracterizar proteínas de unión a c-di-GMP. Esta técnica ha sido previamente informado para ser aplicable a una amplia gama de organismos 1. Aquí se da una descripción detallada del protocolo que utilizamos para investigar tales componentes de señalización. Como un ejemplo, usamos Pseudomonas aeruginosa, un patógeno oportunista en la que c-di-GMP juega un papel crítico en el control de la virulencia y la biopelícula. CCMS identificó 74% (38/51) de los componentes conocidos o previstos de la red de c-di-GMP. Este estudio explica el procedimiento CCMS en detalle, y lo establece como una herramienta potente y versátil para identificar nuevos componentes que participan en la señalización de pequeña molécula.

Para identificar nuevos efectores c-di-GMP en P. aeruginosa se ​​utilizó sistemáticamente CCMS para analizar las fracciones solubles y de membrana de P. aeruginosa cepa PAO1 de un cultivo en fase log (OD 600 = 0,5). Aquí resumimos y discutimos los resultados representativos de esta expedición de pesca. Se utilizaron cuatro réplicas biológicas independientes. Para cada experimento se utilizaron dos concentraciones CDG-CC diferentes (5 mM y 10 mM). Para investigar la especificidad, l...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

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

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.

Captura compuesto Espectrometría de Masas - Una herramienta de gran alcance para identificar nuevos c-di-GMP efectoras Proteínas

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.5K vistas.  Biozentrum of the University of Basel. 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.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI preparación de la muestra: el método de ultra capa fina

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Investigación

Biología

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

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

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 ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Una evaluación cuantitativa de la Lipidome levadura utilizando la espectrometría de masas de ionización por electrospray

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

Split-ubiquitina levadura membrana base de dos híbridos (MITO) del sistema: Una poderosa herramienta para identificar interacciones proteína-proteína

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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 ...

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 ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfiles de metiltransferasas y Otros S-Adenosil- L-Homocisteína proteínas que se unen mediante espectrometría de masas de captura compuesto (MCP)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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 ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

Amida de hidrógeno / deuterio intercambio y MALDI-TOF análisis de espectrometría de masas de Pak2 activación

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Creación de perfiles proteoma redox tiol Uso del etiquetado de espectrometría de masas

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...