Wir möchten Dr. Rich Rogers für die technische Unterstützung danken, mit der Massenspektrometrie analysiert und hilfreiche Diskussionen und Drs. Rob Moritz, Jeff Ranish und Hamid Mirzai für hilfreiche Diskussionen. Diese Arbeit wurde durch die NIH Centers of Excellence finanziert.

Das Wachstum der Zellen und Medien <ol><li> Eine einzelne Kolonie von BY4742Δarg4Δlys1 Zellen über Nacht in 100 mL Rich Media zu einer OD 600 von 1,0 dann Samen in zwei 1-Liter-Kulturen eine minimale Hefemedium (0,17% Yeast Nitrogen Base ohne Ammoniumsulfat oder Aminosäuren, 0,5% Ammoniumsulfat) mit einem volle Ergänzung von Aminosäuren, mit 20 mg / L von isotopisch normalen oder schweren Arginin ergänzt (13 C 6 15 N 4; Isotec) und Lysin (13 C 6 15 N 2; Isotec). </li><li> Die Zellen wurden für 18 Stunden gewachsen, auf eine OD 600 von 1,8. Es ist wichtig, dass die Zellen durch mindestens 9 Generationen gehen auf volle Einbindung der markierten Isotope zu erreichen. Das Licht Probe wurde pelletiert, gewaschen mit sterilem Wasser, reseeded in ein Oleat Medium (isotopisch normalen Arginin und Lysin, 0,2% Oleate (Sigma Chemicals) und 0,5% Tween 40 (Sigma Chemicals)) und stimuliert für weitere 85 Minuten. Daraus ergibt sich eine isotopisch leichte, im Hinblick auf Arginin und Lysin, Probe und einem isotopisch schwere Glukose-grown Referenzprobe Oleat-stimulierte. </li></ol> Cell Lysis, Isolierung und Fraktionierung von Peptiden <ol start="3"><li> Wiegen Zentrifugenflasche. Ernte Proben durch Zentrifugation für 3 Minuten. Entfernen Sie das Medium und saugen überschüssige Flüssigkeit. Wiegen Sie Pellet und Flasche (in der Regel zwischen 1-2 Gramm Ausbeute) blinken dann in flüssigem Stickstoff einfrieren. Fügen Sie ein Volumen entspricht der Pellet-Gewicht von &quot;Schleifen&quot; Puffer, um die gefrorenen Pellets in flüssigem Stickstoff (Phosphate Buffered Saline (PBS, Gibco), 10% Glycerin, Protease-Inhibitoren (SigmaFAST Protease Inhibitor Tabletten, Sigma) und HALT Phosphatase-Inhibitoren (Thermo Scientific )). </li><li> Einfrieren der Mahlbehälter in flüssigem Stickstoff. Warten Sie, bis der flüssige Stickstoff nicht mehr kochen. Übertragen Sie die Pellets zum Schleifen Schiff mit gefrorenem Kugellager. </li><li> Grind bei 600 Umdrehungen pro Minute mit einer 1 min 20 sec-Zyklus mit einer Richtungsumkehr für 3 Minuten. Wieder einfrieren der Mahlbehälter in flüssigem Stickstoff. Wiederholen Sie die 4 mal 15 Minuten insgesamt Mahldauer. </li><li> Sammeln Sie die gefrorenen grindate in ein 50ml Falcon-Röhrchen statt Trockeneis. Bei -80 ° C bis zur Verwendung. </li><li> Machen Sie eine 10ml Lösung von 8 M Harnstoff, 0,1 M Ammoniumhydrogencarbonat, 0,1 M Tris pH 8,6. Add 3 Bände der Harnstoff-Puffer auf 1 Volumen des eingefrorenen grindate (zum Beispiel 3mls der Harnstoff-Puffer hinzugefügt, um einen 1 Gramm Pellet mit 1 ml PBS-Puffer). Unmittelbar beschallen die Mischung mit einer Sondenspitze Sonicator (2 bis 10 Sekunden-Takt). Halten Sie die Spitze in der Nähe der Boden des Röhrchens zu verhindern Schaumbildung. Die grindate sollte sofort in Lösung gehen. </li><li> Deaktivieren Sie das Lysat durch Zentrifugation für 5 Minuten bei 4 ° C. </li><li> Überstand in frische Röhrchen. </li><li> Machen Sie ein frisches Aliquot von 0,5 M Tris [2-carboxy] phosphin (TCEP). In den Probe bei 1:100 für einen 5mm Endkonzentration. Bei 37 ° C für 1 h. </li><li> Alkylierung von reduzierten Cysteinen. Lassen Sie sich auf Raumtemperatur abkühlen. Machen Sie eine frische Aliquot 1M iodoacetamide. Halten Sie in der Dunkelheit (wir wickeln das Rohr in Folie). Hinzufügen, um eine Endkonzentration von 20 mM. Inkubieren im Dunkeln für 1 h bei Raumtemperatur. </li><li> Quench der iodoacetamide mit 20 mM DTT aus einer 1M Lager bei Raumtemperatur für 1 h. </li><li> Quantifizierung des Proteins mit einem Standard-Bradford oder BCA-Assay (nicht beschrieben). Nehmen Sie eine Probe für die Analyse durch SDS-PAGE unten. </li><li> Zur Validierung Einbeziehung der schweren Isotope, verwenden Sie 100 ul des reduzierten und alkylierten Lysat. Verdünnen 1:4 mit dH 2 O, beschallen und fügen Trypsin bei 50:1 Protein Trypsin. Digest für ein Minimum von 4 Stunden, trockenen Probe in einem Speed ​​Vac und dann entsalzt mit einem C18 Ultramicrospin Spalten (The Nest Group). <ol><li> Hydrate Spalte mit 100 ul Acetonitril </li><li> Äquilibrieren mit 200 ul 0,1% TFA. </li><li> Resuspendieren trockenen Probe in 200 ul 0,1% TFA. Legen Sie auf Spalte. Zentrifuge bei ~ 200 xg oder die minimale Geschwindigkeit für den Durchfluss durch die Säule benötigt. </li><li> Zweimal waschen mit 200 ul 0,1% TFA. </li><li> Elution mit zweimal mit 50 ul 60% Acetonitril, 0,1% TFA. </li><li> Dry Probe in einem Speed ​​Vac. </li><li> Resuspendieren Probe in 20 ul 0,1% Ameisensäure. Run 2 ul auf einem Massenspektrometer. Überprüfen Einbeziehung von isotopisch schwere Arginin und Lysin durch eine 10 und 8 Dalton Verschiebung der jeweiligen m / z-Spektren. 98% - Die Aufnahme für jede Aminosäure sollte zwischen 96 sein. </li></ol></li><li> Mix äquivalenten Mengen (mg Protein) des isotopisch schweren und leichten Proben. </li><li> Verdünnte Probe 1:4 mit 20% Methanol. </li><li> Add Trypsin bei 1:200. Inkubieren über Nacht bei 37 ° C. </li><li> Prüfen Sie, ob die Verdauung hat bis zur Fertigstellung durch SDS-PAGE gegangen. Run 2 und 4 ul der Vorverdauung Probe und 8 eind 16 ul der post Trypsin zu verdauen. Visualisieren Sie durch Coomassie-Färbung, die die Verdauung abgeschlossen ist. Wenn es nicht abgeschlossen ist, beschallen die Probe mit einer Sondenspitze Sonicator kurz und zu verdauen wieder mit Trypsin. </li><li> Trocknen Sie die Probe in einem Speed ​​Vac. Nach dem Trocknen mit 200 ul der gefilterten dH 2 O und Wirbel bis das Pellet in Lösung geht. Dry Probe in einem Speed ​​Vac. Resuspendieren wieder mit dH 2 O. Dry Probe wieder. </li><li> Pellet in 0,1% Trifluoressigsäure (TFA; Sigma). Prüfen Sie, ob der pH-Wert um 3 ist mit pH-Streifen. Wenn säuern mit 10% TFA benötigt. </li><li> Pellet aus jedem Niederschlag. </li><li> Desalt die Proben auf einem Waters Sep-Pak Vac 500 mg C18-Säule. Überlasten Sie nicht diese Spalten. Die maximale Menge beträgt 5 mg, aber es ist besser, Last ein bisschen weniger. Teilen Sie die Probe zwischen mehreren Spalten, wenn nötig. <ol><li> Hydrate die Säule mit 5 ml 100% Acetonitril. Zentrifuge bei ~ 200 xg oder die minimale Geschwindigkeit für den Durchfluss durch die Säule benötigt. </li><li> Äquilibrieren Säule mit 5 ml 0,1% TFA. Laden Probe. Sammeln Sie Vor-und Last wieder. </li><li> Waschen mit 5 ml 0,1% TFA. Wiederholen. </li><li> Elution mit 2 ml 60% Acetonitril, 0,1% TFA. </li><li> Dry Probe in einem Speed ​​Vac. </li></ol></li></ol> Peptide Fraktionierung und Isolierung von Phosphopeptide <ol><li> Peptide sind fraktionierte auf HILIC. Wir verwenden eine TOSOH TSK-Gel Amide-80 4,6 mm x 25 cm analytischen Säule mit einer Vorsäule. Nehmen Sie sich Zeit mit diesem Schritt führen Sie die Spalte für 2 Stunden bei 90% A führen Sie dann zwei leere Steigungen vor dem Laden Ihrer Probe. Die maximale Belastung auf einer Säule dieser Größe gedacht, um XX ist, wir in der Regel nicht geladen mehr als 5 mg pro Lauf. </li><li> Solvent A - 98% ACN/0.1% TFA Solvent B - 2% ACN/0.1% TFA Legen Sie in 90% A über 20 &#39;, 90% A bis 85% A über 5&#39;, 85% A bis 60% A über 40 &#39;, 60% A auf 0% A über 5&#39;, 0% A bis 90% A über 5 &#39;. </li><li> Der Durchfluss wird 1ml/min und Fraktionen wurden in 2 Minuten Abständen gesammelt beginnend mit dem 85% A bis 60% A-Gradienten. </li><li> Kombinieren Fraktionen, die Zahl der Fraktionen auf 10 zu reduzieren. Erhöhte Fraktionen wird wahrscheinlich ergeben eine größere Abdeckung des Phosphoproteom. </li></ol> Anreicherung von Phosphopeptide <ol><li> Phosphopeptide angereichert mit PHOS-Select Eisen Affinity Gel. Wir aliquoten unsere IMAC Harz zu vermeiden, wiederholte Einfrieren und Auftauen. Machen Sie 50 ml eines 250 mM Essigsäure, 30% ACN Last Lösung. Bringen Sie den pH-Wert auf 2,7 mit HCl. Resuspendieren Pellets in 500 ul der Last Lösung. Überprüfen pH, passen mit HCl oder NaOH als notwendig. </li><li> Vorwäsche 200 ul IMAC Perlen in einer entsprechenden Spalte. Wir verwenden Molbiotec Spin-Säulen. Add 15 ul einer 50%-Aufschlämmung (Gel zur Lösung Last) zu jeder Fraktion, und inkubieren Sie die Proben für 30 &#39;mit Ende über Ende Rotation. Halten Sie den Fluss für die Analyse. </li><li> Wash Proben 3 mal mit Last-Lösung und einmal mit 500 ul gefiltert dH 2 O. </li><li> Eluieren Peptide mit 2 bis 3 min mit 400 ul 50 mM Na 2 HPO 4 (pH 8,4) Puffer. Transfer in ein Glasgefäß. </li><li> Angesäuert auf pH 3 mit 5 ul 100% ige Ameisensäure, Flash-Freeze in flüssigem Stickstoff und getrocknete Probe in einem Speed ​​Vac. </li><li> Resuspendieren Phosphopeptide in 200 ul 0,1% TFA und C18 sauber wie oben beschrieben. Dry unten eluierten Proben in einem Speed ​​Vac. </li><li> Resuspendieren Peptide in 20 ul 0,1% iger Ameisensäure. Verwenden Sie 2 ul pro Massenspektrometer-Analyse oder nach Bedarf. </li></ol> Hinweise Für viel von diesem Protokoll Sie arbeiten werden &quot;blind&quot;. Die Zell-Lyse kann durch Western-Blot und Bradford oder BCA-Assays und die Trypsinverdauung kann auch leicht beurteilt werden überwacht werden. Die HILIC wird gab eine Spur ähnlich wie diese, oder wie von anderen [1, 2] in Abhängigkeit von der Flüssigchromatographie Maschine, wird gesehen. Die Proben werden mittels Massenspektrometrie überwacht; testen Sie Ihre Proben auf kostengünstigen Maschinen wie LCQs oder Maldis, bevor er zu den hohen Kosten-Analysen auf einem hohen Masse Genauigkeit, hohe Auflösung Maschine. Schließlich, wenn Sie eine Trocknung bis eine Probe mit Säure in ihm, aus Glas verwenden.

<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>

Diese Methode wird zu einer Bereicherung der Phosphopeptide quantitativ analysiert werden kann. Eine Reihe von Parametern können in diesem Protokoll verändert werden, aber der wichtigste Aspekt zu erinnern ist, Ihre Phosphorylierung zu bewahren. Vorbereitung der Zellen für die Quantifizierung kann in einer Reihe von verschiedenen Wegen erreicht werden, zum Beispiel, wenn Sie nicht Etikett Ihre Zellen in vivo, tags wie ICAT [3] oder iTRAC [4] kann nach der Extraktion verwendet werden, um differentiell Label z...

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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>
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quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

Dieses Protokoll beschreibt das Wachstum und die Anregung, mit der Fettsäure Oleat, der isotopisch schweren und leichten S. cerevisiae-Zellen. Die Zellen werden unter Verwendung einer cryolysis Verfahren in einer Kugelmühle Mühle und die daraus resultierenden grindate in Lösung von Harnstoff Solubilisierung gebracht. Dieses Verfahren ermöglicht die Lyse der Zellen in eine metabolisch inaktiven Zustand, Erhaltung Phosphorylierung und verhindert Neuausrichtung der Phosphoproteom während der Zelllyse. Nach der Reduktion, Alkylierung, Trypsinverdauung der Proteine ​​werden die Proben auf C18-Säulen und die Probe Komplexität durch Fraktionierung unter Verwendung von hydrophilen Interaktionschromatographie (HILIC) reduziert entsalzt. HILIC Säulen bevorzugt behalten hydrophile Moleküle, die gut für phosphoproteomics geeignet ist. Phosphorylierte Peptide neigen dazu, später eluieren in der chromatographische Profil als die nicht phosphorylierte Pendants. Nach der Fraktionierung sind Phosphopeptide angereichert mit immobilisierten Metall-Chromatographie, die gegen Gebühr-basierte Affinitäten für Phosphopeptid Bereicherung beruht. Am Ende dieses Verfahrens werden die Proben bereit, quantitativ durch Massenspektrometrie analysiert werden.

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

Beschreibung eines quantitativen Phosphorylierung Verfahren mit cryolysis, Harnstoff solubilziation, HILIC Fraktionierung und IMAC Anreicherung von phosphorylierten Peptiden.

Quantitative Phosphoproteomics in Fatty Acid Stimulated 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 Aufrufe.  Institute for Systems Biology. Beschreibung eines quantitativen Phosphorylierung Verfahren mit cryolysis, Harnstoff solubilziation, HILIC Fraktionierung und IMAC Anreicherung von phosphorylierten Peptiden.

Serie: 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.

Präparation 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 ...

Forschung

Biologie

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.

Identifizierung von Protein-Komplexen mit der quantitativen Proteomik 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

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.

Microarray-Analyse für 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.

Eine Fluoreszenz-Mikroskopie Assay zur Überwachung Mitophagy in der Hefe 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.

Quantifizierung und Analyse der Bildung von HO-Endonuklease Stimulated chromosomalen Translokationen von Single-Strand Annealing 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

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.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung 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

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.

Gekoppelt Assays zur Überwachung Proteinrückfaltung 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

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

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

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

Die Messung der mRNA-Abbau Preise in<em&gt; Saccharomyces cerevisiae</em&gt; Verwenden<em&gt; Rpb1-1</em&gt; Die Stämme

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

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.

Schnelle Identifikation von chemischen Genetische Interaktionen 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. ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Entwicklung Methode Regie in<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Bibliothek Schöpfung und Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...