Name: quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae
Uploaded: 2009-10-12T23:00:00
Description: Watch this Scientific Journal Video about Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae at JoVE.com

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 ...

<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. 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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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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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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 ...

Research

JoVE Journal

Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Dissection of Saccharomyces Cerevisiae Asci

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

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

Microarray Analysis for Saccharomyces cerevisiae

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 ...

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

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

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

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

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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 ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

La medición de mRNA decadencia Tarifas en<em&gt; Saccharomyces cerevisiae</em&gt; Uso<em&gt; Rpb1-1</em&gt; Cepas

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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. ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Determinación de la oxidación de ácidos grasos y la lipogénesis en el ratón hepatocitos primarios

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

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 ...