我々は質量分析をサポートするためUAMSプロテオミクス施設に感謝します。このプロジェクトの資金は、NIHの助成金P20RR015569、P20RR016460とR01DA025755によって提供されました。

このプロトコルは、ブレアらから変更されています。6。 1。 LSD1脱メチル化アッセイ<ol><li> 20μLの最終的なボリュームでは、脱メチル化酵素緩衝液中で0.25μgのジ-メチルヒストンH3ペプチド（ARTKme2QTARKSTGGKAPRKQLYK-ビオチン）と125 ngの組換えLSD1を組み合わせる（50mMのトリス-Cl pH8.5で、50mMのKCl、5mMのMgCl 2、5％グリセロール）。さらに、酵素なしでペプチド単独で制御を行う。コントロールは、セクション3のためであり、還元的メチル化を必要としません。 </li><li> 37℃で2時間インキュベート℃、 </li><li>ペプチドを収集するには、各サンプルに8μLPOROS R2の20ミクロンのビーズを追加し、室温で15分間振とうする。メタノールの1ボリュームとビーズの1ボリュームを停止させることによってPOROSビーズを準備し、次いで、5％ギ酸/ 0.2％TFAの10巻を追加します。 </li><li>条件2の先端に0.1％TFAの20μLを吸引し、ピペットで戻ってそれを押すことにより、C 18 ZipTips。 Tは、0.1％で二回この操作を行う0.1％TFAでFA、再び70％アセトニトリル/ 0.1％TFAで4回、4回。 </li><li> ZipTipsでC 18樹脂の後ろにビーズスラリーをピペッティングし、ピペッターとを介してボリュームを押すことにより、エアコンC 18 ZipTips上に2つのサンプルを（制御および脱メチル化酵素反応）をロードします。 </li><li> 0.1％TFAの20μLで二回ZipTipsを洗ってください。 </li><li> 70％アセトニトリル/ 0.1％TFAの40μLで溶出します。 </li><li> Speed​​Vacコンセントレータと完全にそれぞれの溶離液を凍結乾燥。 </li></ol> 2。還元的メチル化プロトコルのこの部分は、ブレアらから変更されています。とレイメントら 6,7。 <ol><li> 50mMリン酸緩衝液、pH 7.4の100μLの脱メチル化酵素反応を再懸濁する。 </li><li>デメチラーゼ反応に、15 mg / mLのジメチルアミンボランの8μLを追加します。ボランジメチルアミン15 mg / mLのストック溶液を与えるために、50mMリン酸緩衝液、pH7.4中に溶解されています。これは新鮮ななされるべきである。 </li><li>デメチラーゼ反応に、250mMの重水素化ホルムアルデヒドの16μLを追加します。 250 mMの重水素ホルムアルデヒドはH 2 0の株式を希釈することによって調製されています。これは新鮮ななされるべきである。 </li><li> 2時間4℃でインキュベートします。 </li><li>手順2.2、2.3および2.4を繰り返します。 </li><li>手順2.2を繰り返します。 </li><li> 4℃でインキュベートC〜15時間。 </li><li>デメチラーゼサンプルに、還元的メチル化反応をクエンチするために1Mトリス、pH 7.4の12.5μLを追加します。 </li></ol> 3。 MALDIマススペクトロメトリー<ol><li>デメチラーゼ反応にPOROS R2 20ミクロンのビーズを追加し、ステップ1.3から1.6で説明されているようZipTipsに収集します。 </li></ol>注：50 mMリン酸緩衝液、pH 7.4の100μLにコントロールを再中断し、他のサンプルでは、​​ステップ3.1から始まるとして扱う。 <ol start="2"><li> 33％飽和の2μLを用いてMALDIサンプルプレート上にZipTipsから制御および脱メチル化反応のサンプルを溶出させるD 2,5 - ジヒドロキシ安息香酸。 33パーセント2,5 - ジヒドロキシ安息香酸を調製するために、70％アセトニトリル/ 0.1％TFAで飽和33％に希釈し、70％アセトニトリル/ 0.1％TFAで2,5 - ジヒドロキシ安息香酸の飽和​​溶液を作成します。溶出したサンプルが乾燥し結晶化することができます。 </li><li> PerkinElmerSciex MALDI-prOTOF質量分析計または使用可能な高分解能MALDI質量分析計8,9,10を使用してマススペクトルを収集します。 </li><li>スペクトルを見ると質量分析計の製造元から提供されてPerkinElmerSciex TOFworksソフトウェアまたはソフトウェアを使用してピーク面積を抽出します。 </li><li>タンデム質量分析機能を提供可能な質量分析システムとMS 2で反応生成物を確認します。 </li></ol> 4。データ解析<ol><li>重いホルムアルデヒド（図1B）を使用して作成された同位体の重なりを補償するために、ピーク面積の13 C 2と13 C 4同位体relativの（）比を決定還元的メチル化（図2A）を受けていないコントロールサンプルのためのモノアイソトピックピークに電子。 式1：13C2 / 12C = R 1 式2：13C4 / 12C = R 2 これはあなたの実験と質量分析計に特有のこれらの同位体の状態で存在するペプチドの比（R 1とR 2）を与えます。 </li><li>デメチラーゼ反応サンプルの各変更の状態（図2B）に存在するペプチドの相対量を定量化するには、次の式中のR 1とR 2の値を使用します。 式3：H3K4me2 = 1 式4：H3K4me = A 2 - R 1（A 1） 式5：H3K4 = A 3 - R 2（A 1） - [R 1（2 - R 1（A 1））] </li></ol> 5。代表的な結果 LSD1を用いて、定量化するためMassSQUIRMの有効性を示すリジン脱メチル化。などのプロトコルのテキストのセクションで説明したように、我々は、LSD1の125 ngおよび0.25μgのジ - メチルヒストンH3ペプチド（ARTKme2QTARKSTGGKAPRKQLYK-ビオチン）でデメチラーゼアッセイを行った。制御および脱メチル化酵素反応のサンプルは還元的メチル化とMALDI質量分析に供した。還元的メチル化を受けなかったコントロール反応は、このペプチドの典型的な同位体の包絡線を示し、式1および2（図2A）のピーク面積を提供しました。デメチラーゼ反応の質量スペクトルは、式3-5（図2B）のピーク面積を提供した。制御および脱メチル化酵素反応からのピーク面積を用いて、我々は、LSD1は、以下の反応生成物を与えるためにペプチドを脱メチル化することを決定：33.7％のジ - メチルLys4、42.3パーセントモノメチルLys4、24％非メチル化リジン4。 Blair らを参照してください。フルLSD1脱メチル化酵素活性の分析と同様に阻害剤の研究6に。 、酵素などの反応時間などの変数を変更することに注意してください濃度と基質濃度は、アクティビティの詳細な分析が可能になる。 <img alt="図1" src="/files/ftp_upload/3604/3604fig1.jpg" /> 図1。MassSQUIRMの概要。 （A）ヒストンH3のN末端ペプチドは、非（緑）、モノ（赤）、またはジ - （青）のリジン4でメチル化されているとして表示されます。各ペプチドの化学組成の変化が定量化複合体を作る差動電離につながります。 （B）還元メチル化は、すべてのペプチドは、同様にイオン化させ、ジ - メチル状態にすべてのリジン残基に変換します。還元メチル化反応の重ホルムアルデヒドの使用は、オリジナルのメチル化のアイデンティティの保持を可能にします。黒丸が重いメチル化を示しながら、白丸は光メチル化を示しています。Blairら2011年 6 から変更しました 。 <img alt="図2" src="/files/ftp_upload/3604/3604fig2.jpg" /> FigurE 2。MassSQUIRMは差動メチル化ペプチドを定量化するために使用することができます。 （A）ジ-メチル合成ヒストンH3ペプチド（ARTKme2QTARKSTGGKAPRKQLYK-ビオチン）は、モノアイソトピックピークの相対質量とのピーク比を用いて解析した式1および2で、R 1およびR 2として認められた。 （B）と同じ合成ペプチドは、37℃で2時間のためにデメチラーゼバッファ125 ngのLSD1とインキュベートした次いで、試料を分析しMassSQUIRMに供した。重複ピークの混合集団では、図1Bに見つの異なるメチル化状態を表しています。モノアイソトピックピーク下の面積は、1、2、3として認められた。ピーク面積は、式3-5に使用されました。黒丸が重いメチル化を示しながら、白丸は光メチル化を示しています。Blairらから変更しました。 2011 6。

<ol>
	<li>Goldberg, A. D., Allis, C. D., Bernstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+a+landscape+takes+shape.">Epigenetics: a landscape takes shape.</a> Cell. 128, 635-638 (2007).</li><li>Kouzarides, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+modifications+and+their+function.">Chromatin modifications and their function.</a> Cell. 128, 693-705 (2007).</li><li>Blair, L. P., Cao, J., Zou, M. R., Sayegh, J., Yan, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+Regulation+by+Lysine+Demethylase+5+(KDM5)+Enzymes+in+Cancer.">Epigenetic Regulation by Lysine Demethylase 5 (KDM5) Enzymes in Cancer.</a> Cancers (Basel). 3, 1383-1404 (2011).</li><li>Cole, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+probes+for+histone-modifying+enzymes.">Chemical probes for histone-modifying enzymes.</a> Nat. Chem. Biol. 4, 590-597 (2008).</li><li>Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+demethylation+mediated+by+the+nuclear+amine+oxidase+homolog+LSD1.">Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.</a> Cell. 119, 941-953 (2004).</li><li>Blair, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MassSQUIRM:+An+assay+for+quantitative+measurement+of+lysine+demethylase+activity.">MassSQUIRM: An assay for quantitative measurement of lysine demethylase activity.</a> Epigenetics. 6, 490-499 (2011).</li><li>Rayment, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+myosin+subfragment-1:+a+molecular+motor.">Three-dimensional structure of myosin subfragment-1: a molecular motor.</a> Science. 261, 50-508 (1993).</li><li>Collom, S. L., Jamakhandi, A. P., Tackett, A. J., Radominska-Pandya, A., Miller, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYP2E1+active+site+residues+in+substrate+recognition+sequence+5+identified+by+photoaffinity+labeling+and+homology+modeling.">CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling.</a> Arch. Biochem. Biophys. 459, 59-69 (2007).</li><li>Gradolatto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+Yta7+Regulates+Histone+Gene+Expression.">Saccharomyces cerevisiae Yta7 Regulates Histone Gene Expression.</a> Genetics. 179, 291-304 (2008).</li><li>Taverna, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yng1+PHD+finger+binding+to+histone+H3+trimethylated+at+lysine+4+promotes+NuA3+HAT+activity+at+Lysine+14+of+H3+and+transcripiton+at+a+subset+of+targeted+ORFs.">Yng1 PHD finger binding to histone H3 trimethylated at lysine 4 promotes NuA3 HAT activity at Lysine 14 of H3 and transcripiton at a subset of targeted ORFs.</a> Mol. Cell. 24, 785-796 (2006).</li></ol>

我々は、開示することは何もありません。

MassSQUIRMは、モノ - 及びジ - メチル化に関与するリジン脱メチル化酵素活性の包括的な分析のための安価で、定量的な方法です。 MassSQUIRMは、反応生成物のだけでなく、中間体だけでなく、定量化を提供しています。このアッセイは、LSD1や他のヒストン脱メチル化のメカニズムを研究する上で強力なツールとして使用することができます。それはまた、PHF8などの多くの新たに発見されたリジ?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> LSD1 </td><td> BPSサイエンス</td><td> 50100 </td></tr><tr><td> H3K4me2-ビオチンペプチド</td><td>社内で準備</td><td>なし</td></tr><tr><td> POROS R2 20ミクロンのビーズ</td><td>アプライドバイオシステムズ</td><td> 1-1129-06 </td></tr><tr><td> C 18 ZipTip </td><td>ミリポア</td><td> ZTC18M </td></tr><tr><td>トリフルオロ酢酸（TFA） </td><td>サーモ</td><td> 28904 </td></tr><tr><td>アセトニトリル</td><td>フィッシャー</td><td> A996 </td></tr><tr><td> 2,5 - ジヒドロキシ安息香酸</td><td>シグマ</td><td> 85707 </td></tr><tr><td>ボランジメチルアミン</td><td>シグマ</td><td> 180238 </td></tr><tr><td>同位体重いD 2ホルムアルデヒド</td><td>ケンブリッジアイソトープラボラトリーズ</tD&gt; <td> DLM-805から20 </td></tr><tr><td>トリス</td><td>フィッシャー</td><td> BP154 </td></tr><tr><td>塩化カリウム</td><td>フィッシャー</td><td> BP366 </td></tr><tr><td> MgCl 2を </td><td>フィッシャー</td><td> BP214 </td></tr><tr><td>グリセロール</td><td>フィッシャー</td><td> G33 </td></tr><tr><td>ギ酸</td><td>フルカ</td><td> 06440 </td></tr><tr><td>メタノール</td><td>フィッシャー</td><td> A452 </td></tr><tr><td>リン酸ナトリウム</td><td>フィッシャー</td><td> BP329 </td></tr><tr><td> Speed​​Vacコンセントレータ</td><td>学者</td><td> DNA110 </td></tr><tr><td> MALDI-prOTOF質量分析計とTOFworksソフトウェア</td><td> PerkinElmerSciex </td><td>なし</td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

最近では、エピジェネティックな調節因子は1-3さまざまな疾患の主要なプレーヤーとして発見されました。その結果、これらの酵素は、小分子の研究や薬剤開発4の主要な標的である。多くのエピジェネティックなレギュレータは、ごく最近発見されていると分類されているプロセスにまだある。これらの酵素の中ではヒストンや他のタンパク質のリジンからメチル基を除去するリジン脱メチル化酵素である。酵素のこのクラスの新たな性質のために、いくつかのアッセイは、彼らの活動を研究するために開発されている。これは、分類とヒストン脱メチル化酵素のハイスループット研究の両方に道路ブロックされています。現在、非常に少数のデメチラーゼアッセイは存在しています。存在することをそれらは自然の中で定性的な傾向にあると同時に、異なるリジンメチル化の状態（非、モノ - 、ジ - およびトリ - ）の間に識別することはできません。質量分析は、一般的に脱メチル化酵素活性を測定するために使用されるが、現在の質量分析アッセイはないということです差動メチル化ペプチドが異なってイオン化するかどうかに対処できません。メチル化ペプチドの差イオンは、メチル化状態は難しい（図1A）確かに定量的ではないと比較になります。したがって、利用可能なアッセイは、脱メチル化酵素活性の包括的な分析のために最適化されていません。 ここでは、このように本質的に同じ化学種、それらを作るため、イオン化し、すべてのリジンジ - メチル化を強制的に重水素化されたホルムアルデヒドとアミン基の還元的メチル化に基づいていますMassSQUIRMと呼ばれる方法（同位体の還元的メチル化を用いた質量分析の定量）を記述（図1B）と同じです。還元的メチル化後にのみ化学の違いは、MALDIイオン化効率に影響を与えません水素と重水素である。 MassSQUIRMアッセイは非、モノ - 又はジ - メチルリジンの脱メチル化反応生成物に対して特異的である。アッセイはまた、saを与えリジンメチルトランスフェラーゼにも適用可能である私の反応生成物。ここでは、還元的メチル化化学とLSD1の活性を測定するためのMALDI質量分析、合成ペプチドを基板5上に、ジ-およびモノメチル基を除去することができるリジン脱メチル化酵素の組み合わせを使用します。このアッセイは、ラボやプロテオミクス機能を通じてMALDI質量分析計へのアクセス権を持つすべてのラボにシンプルかつ簡単に従順です。アッセイは、〜8倍のダイナミックレンジを持ち、プレートフォーマット5に容易に拡張することができます。

Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

我々は、リジンメチル化の変化を定量化するためにMALDI質量分析法と還元的メチル化化学を使用するための手法を提案する。

リジン脱メチル化酵素活性の定量測定のためのMassSQUIRMのアプリケーション

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

application-masssquirm-for-quantitative-measurements-lysine

Research

JoVE Journal

Biology

13.3K ビュー.  University of Arkansas for Medical Sciences. 我々は、リジンメチル化の変化を定量化するためにMALDI質量分析法と還元的メチル化化学を使用するための手法を提案する。

ビデオ: Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

の活性を測定するアッセイ大腸菌誘導性リジンDecarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

生物学

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

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 ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

フローサイトメトリーによる腎上皮細胞における細胞質ゾルカルシウム測定

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

SPOTペプチド配列を用いてタンパク質リジンメチルトランスフェラーゼの特異性の解析

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

の開発と特性評価<em&gt;インビトロ</em&gt;微小血管ネットワークおよび内皮の定量的測定の[Ca<sup&gt; 2+</sup&gt;]<sub&gt;私</sub&gt;と一酸化窒素の生産

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

電気生理学測定の電気中立運送者の活動を研究への応用

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

部位特異的タンパク質のリジンのアセチル化のサクシニル化化学量論組成データに依存しない取得質量分析法による定量

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.