我々は、技術的な質量分析の解析と有用な議論の支援、そして博士のために博士リッチロジャースに感謝したいと思います。ロブサンモリッツ、ジェフRanish、および有用な議論のためのハミドMirzai。 この作品は、優秀のNIHセンターによって資金を供給された。

細胞増殖とメディア<ol><li>その後、1.0のOD 600〜100mlリッチメディアで一晩BY4742Δarg4Δlys1細胞の単一コロニーを含む最小の酵母培地の二つ1リットルの培養（硫酸アンモニウムまたはアミノ酸を含まない0.17％酵母窒素塩基、0.5％硫酸アンモニウム）にシードとリジン（13 C 6 15 N 2、Isotec）、同位体的に正常または重いアルギニン（Isotec 13 C 6 15 N 4）の20mgの/ Lを添加したアミノ酸の充実した、。 </li><li>細胞は1.8のOD 600に、18時間増殖させた。それは、細胞が標識された同位体の完全な取り込みを実現するために少なくとも9世代を経ることが重要です。光サンプルは、培地を（同位体、通常のアルギニンとリジン、0.2％オレイン酸（シグマケミカル）及び0.5％のTween 40（Sigma社化学））を含むオレイン酸に再シードし、別の85分のために刺激された、滅菌水で洗浄し、ペレット化した。これは、アルギニンやリジン、オレイン酸で刺激したサンプルと同位重いグルコース産の参照試料を基準にして、軽い同位得られます。 </li></ol>ペプチドの細胞の溶解、分離および分画<ol start="3"><li>遠心ボトルを秤量する。 3分間の遠心分離により収穫のサンプル。メディアを取り出し、余分な液体を吸引除去する。ペレットとボトル（通常1〜2グラムの生成します）、液体窒素中で、フラッシュの凍結を量る。液体窒素で凍結ペレット（リン酸塩（PBS、Gibco）を緩衝生理食塩水、10％グリセロール、プロテアーゼ阻害剤（SigmaFASTのプロテアーゼインヒビター錠、シグマ）の&quot;粉砕&quot;バッファのペレットの重量にボリュームと同等を追加し、ホスファターゼ阻害剤をHALT（サーモサイエンティフィック））。 </li><li>液体窒素中で粉砕容器を凍結する。液体窒素が沸騰停止するまで待ちます。冷凍ボールベアリングで、粉砕容器にペレットを転送します。 </li><li> 3分間方向の反転で1分20秒のサイクルで、600rpmで挽く。液体窒素中で粉砕容器を再冷凍。合計粉砕時間15分4回繰り返します。 </li><li> 50ミリリットルファルコンチューブの代わりにドライアイスに冷凍grindateを収集する。使用を-80℃で保存するまで準備。 </li><li> 8M尿素、0.1Mの重炭酸アンモニウム、0.1MトリスpH 8.6の10mlのソリューションとなります。冷凍grindateの1ボリューム（尿素のバッファの例の3mls用PBS緩衝液1mlで1グラムペレットに添加）に尿素緩衝液の3倍量を追加。すぐに、プローブ先端の超音波処理（ - 10秒間のパルス2）との混合物を超音波で分解する。ソリューションの泡立ちを防ぐために、チューブの底近くに先端をキープ。 grindateは直ちにソリューションに行く必要があります。 </li><li> 4℃で5分間、遠心分離によってライセートをクリア℃に</li><li>新鮮なチューブに上清を移します。 </li><li> 0.5Mトリス[2 - カルボキシエチル]ホスフィン（TCEP）の新鮮なアリコートを行う。 5mMの最終濃度のために1:100でサンプルに追加。 37℃を1時間</li><li>削減システインのアルキル化。室温に冷却してください。 1Mヨードアセトアミドの新鮮なアリコートを行う。 （我々は箔で管を包む）暗所で保管してください。 20mmの最終濃度に追加し​​ます。室温で1時間、暗所でインキュベートする。 </li><li> 1時間室温で1M株式から20mm DTTとヨードアセトアミドを癒やす</li><li>標準ブラッドフォードまたはBCAアッセイ（記載されていない）を持つ蛋白質を定量化する。下のSDS PAGEによって分析用のサンプルを取る。 </li><li>重い同位体の取り込みを検証するには、縮小およびアルキル化ライセート100μlを使用してください。のdH 2 Oで1:4に希釈して、超音波で分解し、トリプシンに50:1タンパク質でトリプシンを追加。 C18 Ultramicrospin列（ネストグループ）とVACし、脱塩速度のダウンは最低4時間、乾燥サンプルのダイジェスト。 <ol><li>アセトニトリル100μlの水和物列</li><li> 200μlの0.1％TFAで平衡化する。 </li><li> 200μlの0.1％TFAで乾燥したサンプルを再懸濁します。カラムにロードします。 〜200 XGまたは列内の流れのために必要な最小限の速度で遠心する。 </li><li> 200μlの0.1％TFAで2回洗浄。 </li><li> 50μlの60％アセトニトリル、0.1％TFAで二回で溶出。 </li><li> VACスピードで乾燥したサンプル。 </li><li> 20μlの0.1％ギ酸でサンプルを再懸濁します。質量分析計を2μlを実行します。それぞれm / zのスペクトル10と8ダルトンのシフトによって同位体重いアルギニンとリジンの取り込みを確認してください。 98パーセント - 各アミノ酸のための取り込みは、96の間でなければなりません。 </li></ol></li><li>同位体重いと光の試料の等量（タンパク質のmg）をミックス。 </li><li> 20％メタノールでサンプル1:4希釈する。 </li><li> 1:200でトリプシンを追加。 37℃で一晩インキュベート℃、 </li><li>その消化はSDS PAGEで完了するために行っているを確認してください。 2を実行し、前消化のサンプルの4μLおよび8後トリプシン消化物のdを16μlの。消化が完了したことをクマシー染色によって可視化する。それが完了していない場合、プローブ先端の超音波処理を短時間でサンプルを超音波で分解し、トリプシンで再びダイジェスト。 </li><li> VAC速度でサンプルを下に乾燥させる。乾燥したペレットを溶液になるまでフィルタリングのdH 2 Oと渦の200μlを追加すると。 VAC速度でサンプルを下に乾燥させる。のdH 2 Oで再懸濁します再度サンプルを乾燥させる。 </li><li> 0.1パーセントトリフルオロ酢酸でペレットを再懸濁します（TFA;シグマ）。 pHがストリップを用いて3程度であることを確認してください。 10％のTFAで酸性化する必要があれば。 </li><li>沈殿物からペレット。 </li><li>ウォーターズをSep - Pak Vacを500mgのC18カラムでサンプルを脱塩。これらの列をオーバーロードしないでください。最大量は5 mgのですが、それは少し少なめにロードする方が良いでしょう。必要に応じて複数の列の間にサンプルを分割します。 <ol><li>水和物5ミリリットル、100％アセトニトリルでカラム。 〜200 XGまたは列内の流れのために必要な最小限の速度で遠心する。 </li><li> 5ミリリットルの0.1％TFAでカラムを平衡化する。サンプルをロードします。フローを収集し、再度ロードする。 </li><li> 5ミリリットルの0.1％TFAで洗浄する。繰り返します。 </li><li> 2ミリリットル60％アセトニトリル、0.1％TFAで溶出。 </li><li> VAC速度でサンプルを下に乾燥させる。 </li></ol></li></ol>リン酸化ペプチドのペプチド分画と分離<ol><li>ペプチドは、HILICカラムで分画しています。我々は、東ソーTSK - GELアミド- 80 4.6ミリメートルX 25センチメートルガードカラムと分析カラムが使われています。このステップであなたの時間をかけ、次に実行する2つの空白の勾配は、サンプルをロードする前に、90％で2時間の列を実行します。このサイズはXXになるものと考えられている列の最大負荷は、我々は通常は実行ごとに5つ以上の投与をロードしないでください。 </li><li>溶剤 - 98パーセントACN/0.1％TFA 溶媒B - 2％ACN/0.1％TFA 90％負荷以上20&#39;、90％85％以上の5&#39;、85％〜60％以上40&#39;、60％0％から5以上&quot;、0％〜90％以上5&#39;。 </li><li>流速は1ml/minであり、分画は85％〜60％の勾配で始まる2分間隔で収集した。 </li><li> 10のフラクションの数を減らすためにフラクションを組み合わせる。増加した分画は、おそらくリン酸化プロテオームの高いカバレッジを得られます。 </li></ol>リン酸化ペプチドの濃縮<ol><li>リン酸化ペプチドは、PHOS -セレクト鉄のアフィニティーゲルを用いて濃縮される。我々は、一定分量たちのIMAC樹脂を融解凍結を避けるために。 250mMの酢酸の50mlの、30％ACNの負荷のソリューションを行います。 HClでpH 2.7〜をもたらす。負荷溶液500μlにペレットを再懸濁します。必要に応じてHClまたはNaOHで調整し、pHを確認してください。 </li><li>適切な列のIMACビーズの予備洗浄​​を200μl。我々はMolbiotecスピンカラムを使用してください。 各画分に50％のスラリー（ゲルソリューションをロードするため）の15μlを加え、そして最後の回転を介してエンドと30&#39;​​のサンプルをインキュベートする。分析のための手順を守って下さい。 </li><li>負荷液で試料を3回洗浄し、一回で500μlのフィルタリングのdH 2 Oを</li><li> 2でペプチドを溶出- 50mMののNa 2 HPO 4（pHは8.4）緩衝液400μlの3分。ガラスバイアルに移す。 </li><li> 5μlの100％のギ酸、液体窒素中でフラッシュ凍結、およびVACスピードで乾燥試料ダウンでpH3に酸性化。 </li><li> 0.1％TFA、前述のようにきれいなC18の200μlに再懸濁し、リン酸化ペプチド。 VAC速度で溶出サンプルを乾燥させる。 </li><li> 0.1％ギ酸20μlにペプチドを再懸濁します。質量分析計の分析ごとに2μlを使用するか、または必要に応じて。 </li></ol>ノートずっとこのプロトコルのためには&quot;ブラインド&quot;働くことになります。細胞溶解は、ウェスタンブロットおよびブラッドフォードまたはBCAアッセイによって監視することができる、とトリプシン消化にも容易に評価することができます。 HILICカラムはこれに似たトレースを与えた、または他の[1、2]使用されている液体クロマトグラフィーのマシンに依存することによって見られるだろう。サンプルは、質量分析法によって監視され、そのような高質量精度、高解像度のマシン上で高コストの分析に行く前にLCQsまたはMALDIsなどの安価なマシン上でサンプルをテストします。あなたがそれに酸でサンプルをダウン乾燥最後に場合、ガラスを使用してください。

<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>

このメソッドは、定量的に分析することができるリン酸化ペプチドの濃縮が得られます。パラメータの数は、このプロトコルに変更が、覚えておくべき最も重要な側面は、リン酸化を維持することですすることができます。定量化のための細胞の調製はin vivoであなたの細胞を標識することができない場合など、さまざまな方法で達成することができる、などICATのようなタグ[3]またはITRAC [4]は差定量化のための二つの文化にラベルを付けるためにポスト抽出を使用することができます。目的。 分別のために最も一般的な方法は、SDS PAGE [5、6]、SCXクロマトグラフィー[7]、及びHILICベースのクロマトグラフィー[1、2]によってゲル分画です。我々は、それがグラデーションの終わりまで、ペプチドを保持するため、HILICを選んだのではなくSCXのように、勾配の前方に溶出するが、他のグループは他の方法との良好な成功を収めている。 IMACを使用する際には、洗浄の使用する樹脂の量、インキュベーションの時間あるいは数を増減する必要がある場合があります。また研究は、我々はここで[8]を提示するロードソリューションの初期の化学的性質を変えることで、そこに探している。このメソッドの組み合わせは、95％のリン酸化に60％の間にあるサンプルの分離を許可する必要があります。あなたが特定のシステムのための方法を確立したらあなたは、リン酸化ペプチドの高収率のためのことができる最適化するはずです。 リン酸化ペプチドの濃縮にも酸化チタンと[9、10]またはホスホルアミダイト化学[11、12]を達成することができます、との研究では、それぞれのメソッドはまだリン酸化プロテオーム[13]の別個の部分が重複もたらすことが示されている。

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		<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>
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quantitative phosphoproteomics in fatty acid stimulated saccharomyces cerevisiae

このプロトコルは、同位体の重鎖および軽S.セレビシエ細胞の脂肪酸のオレイン酸、で、成長し、刺激を説明します。細胞は、ボールミルの粉砕機と尿素の可溶化により溶液に持ち込まれた結果grindateで冷凍融解の手順を使用して接地している。この手順では、リン酸化を維持し、細胞溶解中にリン酸化プロテオームの方向転換を防止する、代謝的に不活性状態で細胞の溶解が可能になります。還元、アルキル化、タンパク質のトリプシン消化後、サンプルをC18カラムと親水性相互作用クロマトグラフィー（HILIC）を使用して分別削減サンプルの複雑さに脱塩されています。 HILICカラムには、優先的によくphosphoproteomicsに適した親水性の分子を保持する。リン酸化ペプチドは非リン酸化されたものよりクロマトグラフプロファイルは、後で溶出する傾向がある。分別した後、リン酸化ペプチドはリン酸化ペプチド濃縮用電荷ベースの親和性に依存している固定化金属クロマトグラフィーを、使用して濃縮される。この手順の最後にあるサンプルは、定量的質量分析法により分析する準備が整いました。

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

冷凍融解、尿素solubilziation、HILIC分画とリン酸化ペプチドのIMAC濃縮を用いた定量的リン酸化の手順の説明。

脂肪酸の定量的なPhosphoproteomicsは刺激を受けてサッカロマイセスセレビシエ</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

Universidad San Sebastian

Research

JoVE Journal

Biology

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

10.0K ビュー.  Institute for Systems Biology. 冷凍融解、尿素solubilziation、HILIC分画とリン酸化ペプチドのIMAC濃縮を用いた定量的リン酸化の手順の説明。

ビデオ: 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.

の解剖サッカロマイセスセレビシエ ASCI

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

生物学

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.

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

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.

マイクロアレイ解析のためのサッカロマイセスセレビシエ</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.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

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.

HOエンドヌクレアーゼの形成の定量と分析でアニーリングシングルストランドによる染色体転座を刺激サッカロマイセスセレビシエ</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.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（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.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（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.

における化学遺伝の相互作用の迅速な同定<em&gt;サッカロマイセス·セレビシエ</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).

タンパク質分解中のシクロヘキシミドチェイス分析<em&gt;サッカロマイセス・セレビシエ</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.

の効率的な胞子形成<em&gt;サッカロマイセス・セレビシエ</em&gt; 96マルチウェルフォーマットで

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