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

We willen graag Dr Rich Rogers bedanken voor technische bijstand met massaspectrometrie analyses en behulpzaam discussies en Drs. Rob Moritz, Jeff Ranish, en Hamid Mirzai voor nuttige discussies. Dit werk werd gefinancierd door de NIH Centers of Excellence.

Celgroei en media <ol><li> Een enkele kolonie van BY4742Δarg4Δlys1 cellen &#39;s nachts in 100 ml rijke media om een OD 600 van 1,0 dan zaad in twee 1 liter culturen van een minimale gist medium (0,17% yeast nitrogen base, zonder ammoniumsulfaat of aminozuren, 0,5% ammonium sulfaat) met een volledige aanvulling van aminozuren, aangevuld met 20 mg / L van isotopisch normale of zware arginine (13 C 6 15 N 4, Isotec) en lysine (13 C 6...

<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>

Deze methode levert een verrijking van fosfopeptiden die kwantitatief kunnen worden geanalyseerd. Een aantal parameters kunnen worden gewijzigd in dit protocol, maar het belangrijkste aspect om te onthouden is om het behoud van uw fosforylatie. Voorbereiding van de cellen voor de kwantificering kan worden bereikt in een aantal verschillende manieren, bijvoorbeeld als je niet kunt uw cellen label in vivo, tags zoals ICAT [3] of ITRAC [4] kan gebruikt worden na de winning tot differentieel label twee culturen voo...

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		<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

Dit protocol beschrijft de groei en stimuleren, met het vetzuur oleaat, van isotopisch zware en lichte S. cerevisiae cellen. Cellen worden gemalen met behulp van een cryolysis procedure in een kogelmolen molen en de daaruit voortvloeiende grindate gebracht oplossing van ureum oplosbaar. Deze procedure maakt het mogelijk om lysis van de cellen in een metabool inactieve toestand, met behoud van fosforylatie en het voorkomen van heroriëntatie van de phosphoproteome tijdens de cel lysis. Na de reductie, alkylering, trypsine vertering van de eiwitten, worden de monsters ontzout op C18 kolommen en het monster complexiteit verminderd door fractionering met hydrofiele interactie chromatografie (HILIC). HILIC kolommen bij voorkeur behouden hydrofiele moleculen die goed geschikt is voor phosphoproteomics. Gefosforyleerde peptiden hebben de neiging om later elueren in de chromatografische profiel dan de niet gefosforyleerde tegenhangers. Na fractionering, zijn fosfopeptiden verrijkt met behulp van geïmmobiliseerde metalen chromatografie, die zich baseert op lading op basis van affiniteit voor Phosphopeptide verrijking. Aan het einde van deze procedure de monsters zijn klaar om kwantitatief worden geanalyseerd met behulp van massaspectrometrie.

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

Beschrijving van een kwantitatieve fosforylering procedure met behulp van cryolysis, ureum solubilziation, HILIC fractionering en IMAC verrijking van gefosforyleerde peptiden.

Kwantitatieve Phosphoproteomics in Fatty Acid Gestimuleerd 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 ...

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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Dissection of Saccharomyces Cerevisiae Asci

Dissectie van 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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Identificatie van eiwit-complexen met kwantitatieve 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 ...

Microarray Analysis for Saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

Microarray analyse voor 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

Kwantificering en analyse van de vorming van HO-endonuclease chromosomale translocaties Gestimuleerd door Single-Strand Gloeien in 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

Visualisatie en analyse van mRNA moleculen met behulp van fluorescentie<em&gt; In Situ</em&gt; Hybridisatie in<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

Gekoppeld Testen voor Monitoring Protein Refolding in<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

Meting van mRNA Decay tarieven in<em&gt; Saccharomyces cerevisiae</em&gt; Het gebruik van<em&gt; Rpb1-1</em&gt; Stammen

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

Snelle identificatie van chemische Genetische Interacties in<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

Bepaling van vetzuuroxidatie en Lipogenese in Mouse primaire hepatocyten

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

Cycloheximide Chase Analyse van de afbraak van eiwitten in<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 ...