Nos gustaría dar las gracias al Dr. Rich Rogers para la asistencia técnica con análisis de espectrometría de masas y sus útiles comentarios, y los Dres. Rob Moritz, Ranish Jeff, y Hamid Mirzai útil para los debates. Este trabajo fue financiado por el NIH Centros de Excelencia.

Crecimiento de las células y los medios de comunicación <ol><li> Una sola colonia de BY4742Δarg4Δlys1 células durante la noche en 100 ml de medio rico para un OD 600 de 1,0 después de semillas en dos litros de una cultura medio levadura mínima (0,17% de levadura base de nitrógeno, sin sulfato de amonio o aminoácidos, 0,5% de sulfato de amonio) que contiene una complemento completo de aminoácidos, complementada con 20 mg / L de la arginina normal o isótopos pesados ​​(13 C 6 15 N 4; Isotec) y lisina (13 C 6 15 N 2; Isotec). </li><li> Las células fueron cultivadas durante 18 horas, a una OD 600 de 1,8. Es muy importante que las células pasan por lo menos 9 generaciones para lograr la plena incorporación de los isótopos marcados. La muestra de la luz se sedimentaron, se lava con agua estéril, resembrado en un medio que contiene oleato (isótopos normales arginina y la lisina, un 0,2% Oleato (Sigma Chemicals) y el 0,5% de Tween 40 (Sigma Chemicals)) y estimulada por otros 85 minutos. El resultado es un isotópicamente ligero, con respecto a la arginina y la lisina, oleato estimulada por la muestra y una isótopos pesados ​​de glucosa crecido muestra de referencia. </li></ol> Lisis celular, aislamiento y fraccionamiento de los péptidos <ol start="3"><li> Pesar botella de centrífuga. Muestras de la cosecha por centrifugación durante 3 minutos. Eliminar los medios de comunicación y aspirar el exceso de líquido. Pesar de pellets y la botella (por lo general producirá entre 2.1 gramos) y luego congelar flash en nitrógeno líquido. Añadir un volumen equivalente al peso del pellet &#39;rectificado&#39; buffer a los sedimentos congelados en nitrógeno líquido (tampón fosfato salino (PBS, Gibco), 10% de glicerol, inhibidores de la proteasa (SigmaFAST comprimidos inhibidor de la proteasa, Sigma) y inhibidores de la fosfatasa HALT (Thermo Scientific )). </li><li> Congelar el recipiente de molienda en nitrógeno líquido. Espere hasta que el nitrógeno líquido deja hervir. La transferencia de la pastilla a la nave de molienda, con rodamientos de bolas congeladas. </li><li> Molienda a 600 rpm con un 1 minuto 20 segundos con un ciclo de inversión de la dirección durante 3 minutos. Vuelva a congelar el recipiente de molienda en nitrógeno líquido. Repetir cuatro veces más de 15 minutos de tiempo total de molienda. </li><li> Recoger el grindate congelado en un hielo de 50 ml Falcon tubo lugar seco. Almacenar a -80 ° C hasta su utilización. </li><li> Prepare una solución de 10 ml de urea 8 M, bicarbonato de amonio 0,1 M, 0,1 M Tris pH 8,6. Añadir 3 volúmenes de tampón de urea a un volumen de la grindate congelados (por ejemplo 3mls del buffer de urea añade una pastilla de 1 gramo con 1 ml de tampón PBS). Inmediatamente sonicar la mezcla con un baño de ultrasonidos punta de la sonda (2 - 10 pulsos de segundo). Mantener la punta en la parte inferior del tubo para evitar la formación de espuma de la solución. El grindate debe ir inmediatamente a la solución. </li><li> Borrar el lisado por centrifugación durante 5 minutos a 4 ° C. </li><li> Transferir el sobrenadante a tubos nuevos. </li><li> Hacer una alícuota de 0,5 M Tris [2-carboxietil] fosfina (TCEP). Añadir a la muestra a 1:100 para una concentración final de 5 mm. Se incuba a 37 ° C durante 1 h. </li><li> Alquilación de cisteína reducidos. Dejar enfriar a temperatura ambiente. Hacer una parte alícuota de la yodoacetamida 1M. Mantener en la oscuridad (que envuelve el tubo en papel de aluminio). Añadir a una concentración final de 20 mM. Incubar en la oscuridad durante 1 hora a temperatura ambiente. </li><li> Apagar la yodoacetamida con 20 mM DTT de una reserva de 1 millón a temperatura ambiente durante 1 h. </li><li> Cuantificar la proteína de Bradford con un estándar o ensayo de BCA (no se describe). Tomar una muestra para su análisis por SDS-PAGE a continuación. </li><li> Para validar la incorporación de los isótopos pesados, utilice 100 l de lisado reducida y alquilada. Diluir 1:4 con dH 2 O, ultrasonidos y añadir tripsina a 50:1 proteína a la tripsina. De digerir para un mínimo de 4 horas, muestra seca en un Speed ​​Vac y desalar luego con un Ultramicrospin columnas C18 (El Grupo de nido). <ol><li> Columna de hidratarse con 100 l de acetonitrilo </li><li> Equilibre con 200 l TFA 0,1%. </li><li> Resuspender la muestra seca en 200 l TFA 0,1%. De carga sobre la columna. Centrifugar a 200 xg ~ o de la velocidad mínima necesaria para el flujo a través de la columna. </li><li> Lavar dos veces con 200 l TFA 0,1%. </li><li> Eluir con dos veces con 50 l de acetonitrilo 60%, 0,1% de TFA. </li><li> Muestra seca en un Speed ​​Vac. </li><li> Resuspender la muestra en 20 l de ácido fórmico al 0,1%. Ejecutar 2 l de un espectrómetro de masas. Compruebe la incorporación de la arginina y la lisina isótopos pesados ​​por un turno de 10 y 8 Dalton en los respectivos m / z espectros. La incorporación de cada aminoácido debe estar entre 96 a 98%. </li></ol></li><li> Mezcle cantidades equivalentes (mg de proteínas) de las muestras de isótopos pesados ​​y ligeros. </li><li> Diluir la muestra 1:4 con 20% de metanol. </li><li> Añadir a la tripsina 1:200. Incubar toda la noche a 37 ° C. </li><li> Compruebe que la digestión se ha ido hasta el final por SDS-PAGE. De ejecución 2 y 4 l de la muestra predigestión y un 8d 16 l de la digestión después de la tripsina. Visualizar por tinción Coomassie que la digestión es completa. Si no está completo, sonicar la muestra con un breve sonda sonicador punta y digerir de nuevo con tripsina. </li><li> Secar la muestra en un Speed ​​Vac. Cuando se seca añadir 200 l de filtrado dH 2 O y agitar hasta que el precipitado se disuelve. Muestra seca en un Speed ​​Vac. Volver a suspender de nuevo con dH 2 O. Muestra seca de nuevo. </li><li> Resuspender el pellet en 0,1% de ácido trifluoroacético (TFA, Sigma). Comprobar que el pH es de alrededor de 3 con tiras de pH. Si es necesario acidificar con 10% de TFA. </li><li> Pellet cualquier precipitado. </li><li> Desalinizar las muestras en una Waters Sep-Pak Vac 500 mg columna C18. No sobrecargue las columnas. La cantidad máxima es de 5 mg, pero es mejor para cargar un poco menos. Dividir la muestra entre varias columnas si es necesario. <ol><li> Hidratar la columna con 5 ml de acetonitrilo al 100%. Centrifugar a 200 xg ~ o de la velocidad mínima necesaria para el flujo a través de la columna. </li><li> Equilibrar la columna con 5 ml de TFA 0,1%. Carga de la muestra. Recoge el flujo y la carga de nuevo. </li><li> Lavar con 5 ml de TFA 0,1%. Repetir. </li><li> Eluir con 2 ml de acetonitrilo al 60%, 0,1% de TFA. </li><li> Muestra seca en un Speed ​​Vac. </li></ol></li></ol> Fraccionamiento de péptidos y aislamiento de fosfopéptidos <ol><li> Los péptidos son fraccionados en una columna HILIC. Utilizamos un TOSOH TSK-Gel Amida-80 4,6 mm x 25 cm columna analítica con una columna de seguridad. Tómese su tiempo con este paso, ejecute la columna durante 2 horas a 90% A continuación, ejecute dos gradientes en blanco antes de cargar la muestra. La carga máxima de una columna se considera que el tamaño en alrededor de XX, por lo general no se carga más de 5 mg por ciclo. </li><li> Disolvente A - 98% ACN/0.1% TFA Disolvente B - 2% ACN/0.1% TFA De carga en un 90% más de 20 &#39;, 90% A 85% A más de 5&#39;, un 85% al ​​60% más de 40 &#39;, 60% A 0% A más de 5, 0% a un 90% más de una 5 &#39;. </li><li> El caudal es 1ml/min y las fracciones se recogieron a intervalos de 2 minutos a partir de el 85% a 60% A un gradiente. </li><li> Combinar fracciones de reducir el número de fracciones a 10. El aumento de las fracciones es probable que el rendimiento mayor cobertura de la phosphoproteome. </li></ol> Enriquecimiento de fosfopéptidos <ol><li> Fosfopéptidos se enriquecen con fosfato de hierro Seleccione gel de afinidad. Nos alícuota nuestra resina IMAC para evitar congelamiento repetido descongelación. Hacer 50 ml de ácido acético 250 mM, 30% de solución de carga de ACN. Llevar el pH a 2.7 con HCl. Volver a suspender las pastillas en 500 l de solución de carga. Verifique el pH, ajustar con HCl o NaOH según sea necesario. </li><li> Prelavado 200 l de bolas de IMAC en una columna adecuada. Nosotros usamos las columnas Molbiotec giro. Añadir 15 l de una mezcla del 50% (gel para cargar la solución) a cada fracción, y se incuban las muestras durante 30 &#39;con el extremo final sobre la rotación. Mantener el flujo para el análisis. </li><li> Lavar las muestras 3 veces con solución de carga y una vez con 500 l filtrado dH 2 O. </li><li> Péptidos eluir con 2 - 3 min con 400 l de 50 mM Na 2 HPO 4 (pH 8,4) de amortiguación. Transferir a un frasco de vidrio. </li><li> Acidificar a pH 3 con ácido 5-l fórmico al 100%, la congelación de flash en nitrógeno líquido, y la muestra seca en un Speed ​​Vac. </li><li> Fosfopéptidos Resuspender en 200 l de 0,1% de TFA y C18 limpia como se describe anteriormente. Secan las muestras eluidas en un Speed ​​Vac. </li><li> Resuspender péptidos en 20 l de 0,1% de ácido fórmico. Use 2 l por el análisis de espectrometría de masas o como sea necesario. </li></ol> Notas Durante gran parte de este protocolo que va a trabajar &quot;a ciegas&quot;. La lisis celular puede ser controlado por el Western Blot y ensayos de Bradford o BCA, y la digestión de la tripsina también se puede evaluar fácilmente. La columna HILIC se dio una huella similar a esta, o como se ve por otros [1, 2] en función de la máquina de cromatografía de líquidos que se utiliza. Las muestras son supervisados ​​por espectrometría de masas, las pruebas de las muestras en las máquinas de bajo costo, como LCQs o Maldis antes de ir a los análisis de costos de alta precisión en una gran masa, máquina de alta resolución. Por último, si un secado por una muestra con ácido en el mismo, el uso de cristal.

<ol>
	<li>Albuquerque, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multidimensional+chromatography+technology+for+in-depth+phosphoproteome+analysis.">A multidimensional chromatography technology for in-depth phosphoproteome analysis.</a> Mol Cell Proteomics. 7 (7), 1389-1396 (2008).</li><li>McNulty, D. E., Annan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography+reduces+the+complexity+of+the+phosphoproteome+and+improves+global+phosphopeptide+isolation+and+detection.">Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.</a> Mol Cell Proteomics. 7 (5), 971-980 (2008).</li><li>Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+complex+protein+mixtures+using+isotope-coded+affinity+tags.">Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.</a> Nat Biotechnol. 17 (10), 994-999 (1999).</li><li>Ross, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Gruhler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteomics+applied+to+the+yeast+pheromone+signaling+pathway.">Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.</a> Mol Cell Proteomics. 4 (3), 310-327 (2005).</li><li>Wilson-Grady, J. T., Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoproteome+analysis+of+fission+yeast.">Phosphoproteome analysis of fission yeast.</a> J Proteome Res. 7 (3), 1088-1097 (2008).</li><li>Villen, J., Gygi, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+SCX/IMAC+enrichment+approach+for+global+phosphorylation+analysis+by+mass+spectrometry.">The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.</a> Nat Protoc. 3 (10), 1630-1638 (2008).</li><li>Jensen, S. S., Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+some+experimental+procedures+on+different+phosphopeptide+enrichment+techniques.">Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.</a> Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).</li><li>Larsen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+selective+enrichment+of+phosphorylated+peptides+from+peptide+mixtures+using+titanium+dioxide+microcolumns.">Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.</a> Mol Cell Proteomics. 4 (7), 873-886 (2005).</li><li>Pinkse, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+isolation+at+the+femtomole+level+of+phosphopeptides+from+proteolytic+digests+using+2D-NanoLC-ESI-MS/MS+and+titanium+oxide+precolumns.">Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.</a> Anal Chem. 76 (14), 3935-3943 (2004).</li><li>Tao, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+phosphoproteome+analysis+using+a+dendrimer+conjugation+chemistry+and+tandem+mass+spectrometry.">Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry.</a> Nat Methods. 2 (8), 591-598 (2005).</li><li>Zhou, H., Watts, J. D., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+approach+to+the+analysis+of+protein+phosphorylation.">A systematic approach to the analysis of protein phosphorylation.</a> Nat Biotechnol. 19 (4), 375-378 (2001).</li><li>Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+isolation+of+distinct,+overlapping+segments+of+the+phosphoproteome.">Reproducible isolation of distinct, overlapping segments of the phosphoproteome.</a> Nat Methods. 4 (3), 231-237 (2007).</li></ol>

Este método producirá un enriquecimiento de fosfopéptidos que puede ser analizada cuantitativamente. Una serie de parámetros pueden ser alterados en este protocolo, pero el aspecto más importante a recordar es el de preservar su fosforilación. Preparación de las células para la cuantificación se puede lograr de varias maneras diferentes, por ejemplo, si no se puede etiquetar las células in vivo, las etiquetas, como ICAT [3] o iTRAC [4] se puede utilizar después de la extracción de etiquetar diferenc...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Isotec&trade; L-Arginine-13C6,15N4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>608033</td>
<td>&nbsp;</td>
<tr>
<td>Isotec&trade; L-Lysine-15N2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>609021</td>
<td>&nbsp;</td>
<tr>
<td>GIBCO&#x2122; Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>70011044</td>
<td>&nbsp;</td>
<tr>
<td>SigmaFAST Protease Inhibitor Tablets</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Pierce Halt Phosphatase Inhibitor Cocktail</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>78420</td>
<td>&nbsp;</td>
<tr>
<td>Retsch PM100 Ball Mill Grinder </td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>20.540.0001</td>
<td>&nbsp;</td>
<tr>
<td>Retsch 125 ml Stainless Steel Grinding Jar</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>01.462.0148</td>
<td>&nbsp;</td>
<tr>
<td>Ultramicrospin C18 column</td>
<td>&nbsp;</td>
<td>The Nest Group</td>
<td>SUM SS10</td>
<td>&nbsp;</td>
<tr>
<td>Sep-Pak Vac 500 mg C18</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT043395</td>
<td>&nbsp;</td>
<tr>
<td>TSK-Gel Amide-80 4.6 mm x 25 cm analytical column</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>13071</td>
<td>This is the HILIC chromatographic column.</td>
</tr>
<tr>
<td>Guard column 4.6 mm ID x1 cm</td>
<td>&nbsp;</td>
<td>TOSOH BioSciences</td>
<td>19021</td>
<td>We recommend this guard column to extend the life of your HILIC column.</td>
</tr>
<tr>
<td>PHOS-Select&trade; Iron Affinity Gel</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P9740</td>
<td>This is the IMAC resin. Aliquot and store.</td>
</tr>		</tbody>
		</table>
		</div>

quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

Este protocolo describe el crecimiento y la estimulación, con el oleato de ácidos grasos, de las células de Saccharomyces isótopos pesados ​​y ligeros S.. Las células son de tierra mediante un procedimiento cryolysis en un molinillo de molino de bolas y el grindate resultante llevó a la solución de solubilización urea. Este procedimiento permite la lisis de las células en un estado metabólicamente inactiva, la preservación de la fosforilación y la prevención de la reorientación de la phosphoproteome durante la lisis celular. Tras la reducción, alquilación, digestión con tripsina de las proteínas, las muestras se desala en las columnas C18 y la complejidad de la muestra reducida por fraccionamiento utilizando cromatografía de interacción hidrofílica (HILIC). Columnas HILIC preferentemente retener moléculas hidrofílicas, que es muy adecuado para phosphoproteomics. Péptidos fosforilados suelen eluir más adelante en el perfil cromatográfico de las contrapartes no fosforilada. Después de fraccionamiento, fosfopéptidos se enriquecen mediante cromatografía de inmovilizado de metal, que se basa en la base de carga para el enriquecimiento de fosfopéptidos afinidades. Al final de este procedimiento las muestras están listos para ser analizada cuantitativamente por espectrometría de masas.

Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Descripción de un procedimiento de fosforilación cuantitativos utilizando cryolysis, solubilziation urea, fraccionamiento HILIC y enriquecimiento IMAC de péptidos fosforilados.

Phosphoproteomics cuantitativo de ácidos grasos estimulada Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

quantitative-phosphoproteomics-fatty-acid-stimulated-saccharomyces

Research

JoVE Journal

Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K vistas.  Institute for Systems Biology. Descripción de un procedimiento de fosforilación cuantitativos utilizando cryolysis, solubilziation urea, fraccionamiento HILIC y enriquecimiento IMAC de péptidos fosforilados.

Video: Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

La disección de Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Investigación

Biología

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 ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

El análisis de microarrays para Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every eukaryotic genome. This process is an important mechanism for generating diversity between and within organisms1-3. The human genome consists of approximately 40% repetitive sequence of retrotransposon origin, including a variety of LINEs and SINEs4. Exchange events between these repetitive elements can lead to genome rearrangements, including translocations, that can disrupt gene dosage and expression that can result in autoimmune and cardiovascular diseases5, as well as cancer in humans6-9.
Exchange between repetitive elements occurs in a variety of ways. Exchange between sequences that share perfect (or near-perfect) homology occurs by a process called homologous recombination (HR). By contrast, non-homologous end joining (NHEJ) uses little-or-no sequence homology for exchange10,11. The primary purpose of HR, in mitotic cells, is to repair double-strand breaks (DSBs) generated endogenously by aberrant DNA replication and oxidative lesions, or by exposure to ionizing radiation (IR), and other exogenous DNA damaging agents. 
In the assay described here, DSBs are simultaneously created bordering recombination substrates at two different chromosomal loci in diploid cells by a galactose-inducible HO-endonuclease (Figure 1). The repair of the broken chromosomes generates chromosomal translocations by single strand annealing (SSA), a process where homologous sequences adjacent to the chromosome ends are covalently joined subsequent to annealing. One of the substrates, his3-&Delta;3', contains a 3' truncated HIS3 allele and is located on one copy of chromosome XV at the native HIS3 locus. The second substrate, his3-&Delta;5', is located at the LEU2 locus on one copy of chromosome III, and contains a 5' truncated HIS3 allele. Both substrates are flanked by a HO endonuclease recognition site that can be targeted for incision by HO-endonuclease. HO endonuclease recognition sites native to the MAT locus, on both copies of chromosome III, have been deleted in all strains. This prevents interaction between the recombination substrates and other broken chromosome ends from interfering in the assay. The KAN-MX-marked galactose-inducible HO endonuclease expression cassette is inserted at the TRP1 locus on chromosome IV. The substrates share 311 bp or 60 bp of the HIS3 coding sequence that can be used by the HR machinery for repair by SSA. Cells that use these substrates to repair broken chromosomes by HR form an intact HIS3 allele and a tXV::III chromosomal translocation that can be selected for by the ability to grow on medium lacking histidine (Figure 2A). Translocation frequency by HR is calculated by dividing the number of histidine prototrophic colonies that arise on selective medium by the total number of viable cells that arise after plating appropriate dilutions onto non-selective medium (Figure 2B). A variety of DNA repair mutants have been used to study the genetic control of translocation formation by SSA using this system12-14.

Quantitation and Analysis of the Formation of HO-Endonuclease Stimulated Chromosomal Translocations by Single-Strand Annealing in Saccharomyces cerevisiae

The HO-stimulated translocation assay monitors single-strand annealing following the creation of DNA double-strand breaks at multiple loci in diploid Saccharomyces cerevisiae. This mechanism may model genome rearrangements in somatic cells of higher eukaryotes following exposure to high doses of ionizing radiation.

Cuantificación y análisis de la formación de HO-endonucleasa Estimulado translocaciones cromosómicas por un solo capítulo recocido en Saccharomyces cerevisiae</em

Genetic variation is frequently mediated by genomic rearrangements that arise through interaction between dispersed repetitive elements present in every ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

La rápida identificación de químicos interacciones genéticas en<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Análisis cicloheximida persecución de degradación de proteínas en<em&gt; Saccharomyces cerevisiae</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

La esporulación eficiente de<em&gt; Saccharomyces cerevisiae</em&gt; En un formato de 96 multipocillos

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

Phosphoproteomics cuantitativo de ácidos grasos estimulada Saccharomyces cerevisiae

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