この研究は、もともとモル細胞プロテオミクスに掲載されました。 soldoの複数形M.およびクロマチン機能ドメインのBonaldi T.ザ·プロテオミクス研究は、異なるヘテロクロマチンコンポーネントのMCPの中で小説相乗作用を明らかにしている。 2013; 12：64-80。 ©生化学分子生物学のためのアメリカの社会。私たちは、原稿の重要な読書のためのロベルタNoberini（技術とIEOのイタリアの研究所、イタリア）に感謝。結核作業はジョバンニArmenise ​​ - ハーバード財団キャリア開発プログラム、がん研究のためのイタリアの協会と厚生労働省のイタリアからの補助金によってサポートされています。 MSの作業はFIRCフェローシップによってサポートされていました。

<ol>
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利害の対立が宣言されていません。

我々は最近ChroP、クロマチンのタンパク質成分の大規模な特性評価のための定量的な戦略を記載している。 ChroPは、その強度から利益とそれぞれの限界を克服し、エピジェネティックなフィールドは、チップとMSで使用される2つの相補的なアプローチを組み合わせています。深いシーケンシング（チップ配列）に結合されたチップは、数ヌクレオソーム35の解像度でヒストン修飾のゲ?...

クロマチンはすべてのDNA媒介プロセスの主要なテンプレートとして関与している非常に動的な核タンパク質複合体である。ヌクレオソームはクロマチンの基本的な繰り返し単位であり、DNAの147塩基対が、1,2ラップ回される各カノニカルヒストンH2A、H2B、H3及びH4、2分子を含有するタンパク質性八量体コアからなる。すべてのコアヒストンは、ヌクレオソームの外に突出している球状ドメインで柔軟なN末端 ​​「テール」として構成されている。クロマチン構造とダイナミクスを調節するための主要なメカニズムの一つは、主にヒストン3,4のN-末端に生じる共有結合性翻訳後修飾（PTMを）に基づいている。ヒストン修飾がヒストン-DNA間、またはヌクレオソーム間の接触を変化させることにより、高次クロマチン構造を変化させるため、DNA結合タンパク質（ シスメカニズム）のアクセスを制御することのいずれかによって機能することができ、又はdockinとして作用することにより調節タンパク質のために、単一のユニット、または多量体複合体に埋め込まれたようなグラム·サイト。そのような調節タンパク質は、異なる方法でその機能を発揮することができる：直接遺伝子発現（ すなわち、TAFタンパク質）を調節することによって、またはヌクレオソーム位置決め（ すなわち、クロマチンリモデリング複合体）を変えることにより、又は他のヒストン残基を修飾することにより（メチルトランスフェラーゼまたはアセチルを有する、すなわちタンパク質を転移酵素活性）（ トランスメカニズム）5。特定のクロマチン座で明確なPTMパターンクラスタは異なる部位で異なる修正が基本となるDNAの機能状態を仲介する分子コードを生成するために相乗作用がありという仮説の精緻化につながっていることを観察。 「ヒストンコード仮説は、「年間で大規模なコンセンサスを得ていますが、その実験的検証は技術的な限界6,7によって戻って開催されました。 質量分析（MS）ベースのプロテオミクスとして浮上しているヒストン修飾パターンをマッピングすると、クロマチン結合タンパク質8を特徴づけるための強力なツール。 MSは、ペプチドの実験と理論質量との間の特定のΔmassとして変更を検出します。個々のヒストンのレベルでは、MSは、それらの9月14日の間で新たな修正解きほぐすinterplaysの検出を可能に、PTMをマッピングする公平かつ包括的な方法を提供する。 近年、多くの戦略は、無傷の有糸分裂染色体15、可溶性hPTM結合タンパク質16-18の同定および特定のクロマチン領域の単離および分析の特徴付けを含む、クロマチンのプロテオミクス組成を分析するために開発されている（ すなわちテロメア）19,20。しかし、ヒストンPTMを、変異体、およびクロマチン関連タンパク質間の遺伝子座特異的相乗効果の調査はまだ不完全です。ここでは、（ChroPという名前の新しいアプローチを説明します我々は効率的に機能的に異なるクロマチンドメインを特徴づけるために開発されたことをCHRO MATIN のP roteomics）21。このアプローチは、濃縮されたサンプルの効率的なMSベースのプロテオミクス解析のために、クロマチン免疫沈降（チップ）、エピジェネティック研究に使用される十分に確立されたプロトコルを適応させる。我々は、入力およびMSによって対処かごとして用いるクロマチンの種類に応じて、2つの異なるプロトコルを開発した。特に：1）MNaseで消化した未定着ネイティブクロマチンのチップは、モノヌクレオソームを精製するとCO会合hPTMs（N-ChroP）を解剖するために採用されている。 2）超音波処理によって断片化、架橋クロマチンのチップは、タンパク質（X-ChroP）を結合すべての共同豊かクロマチンを特徴づけるSILACベースinteractomics戦略と組み合わせて使用​​されている。ここでは、免疫沈降ステップのための餌としてのH3K9me3を使用して、ヘテロクロマチンを豊かにし、研究するための、N-とX-ChroPの組み合わせを示している。 ChroPの使用が拡張することができるこのようにエピジェネティクスでの様々なアプリケーションへの道を切り開いて、明確なクロマチン上の領域、または別の機能的状態への移行時に、同じ領域内のクロマチン組成の変化のどちらかを研究する。

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">DMEM&#160;</td>
			<td style="width:145px;">Lonza</td>
			<td style="width:119px;">BE12-614F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">FBS</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:119px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">SILAC DMEM</td>
			<td style="width:145px;">M-Medical</td>
			<td style="width:119px;">FA30E15086</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Dialyzed FBS</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:119px;">26400-044</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Lysine 0 (12C6 14N2 L-lysine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">L8662</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Arginine 0 (12C6 14N4 L-arginine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">A6969</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lysine 8 (13C6 15N2 L-lysine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td>68041</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Arginine 10 (13C6 15N4 L-arginine)</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td>608033</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Micrococcal Nuclease</td>
			<td style="width:145px;">Roche</td>
			<td style="width:119px;">10 107 921 001</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:271px;">Complete, EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td style="width:145px;">Roche</td>
			<td style="width:119px;">04 693 132 001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length</td>
			<td style="width:145px;">Spectrumlabs</td>
			<td style="width:119px;">132720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">QIAquick PCR purification kit</td>
			<td style="width:145px;">QIAGEN</td>
			<td style="width:119px;">28104</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Anti-Histone H3 tri-methylated K9-ChIP grade</td>
			<td>Abcam</td>
			<td>ab8898</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Histone H3 peptide tri-methyl K9&#160;</td>
			<td>Abcam</td>
			<td style="width:119px;">ab1773</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td style="width:119px;">100.04D</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">NuPAGE Novex 4-12%&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Bis-Tris Gel&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>NP0335BOX</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Colloidal Blue Staining Kit</td>
			<td>Invitrogen&#160;</td>
			<td>LC6025</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LDS Sample Buffer</td>
			<td>Invitrogen&#160;</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Formaldheyde</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Aceti anhydride-d6</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">175641-1G</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Formic Acid</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">94318-50ML-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Iodoacetamide &#8805;99% (HPLC), crystalline</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">I6125</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">DL-Dithiothreitol</td>
			<td style="width:145px;">Sigma Aldrich</td>
			<td style="width:119px;">43815</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Sequencing Grade Modified Trypsin, Frozen 100ug (5 &#215; 20&#956;g)</td>
			<td>Promega</td>
			<td style="width:119px;">V5113</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Nanospray OD 360&#956;m x ID 75&#956;m, tips ID 8&#956;m uncoated Pk 5</td>
			<td style="width:145px;">Microcolumn Srl</td>
			<td style="width:119px;">FS360-75-8-N-5-C15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">&#160; ReproSil-Pur 120 C18-AQ, 3 &#181;m&#160;&#160; 15 % C</td>
			<td style="width:145px;">Dr. Maisch GmbH</td>
			<td style="width:119px;">r13.aq.</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">C18 extraction disk, 47 mm</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">1&#8203;2&#8203;1&#8203;4&#8203;5&#8203;0&#8203;0&#8203;4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Carbon extraction disk, 47 mm</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">12145040</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">Cation extraction disk</td>
			<td style="width:145px;">Agilent Technologies</td>
			<td style="width:119px;">66889-U</td>
			<td></td>
		</tr>
	</tbody>
</table>

the chrop approach combines chip and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin

クロマチンは様々なDNA依存性プロセスを制御するDNAやタンパク質で作られた非常にダイナミックな核タンパク質複合体である。特定の領域におけるクロマチン構造および機能は、転写因子、およびDNAメチル化を含むヒストンの翻訳後修飾（hPTMs）および変異体、クロマチン結合タンパク質の局所的な濃縮によって調節される。個別の機能領域でのクロマチン組成のプロテオミクス特性評価は、これまで質量分析（MS）によるその後の詳細な分析のための適切な純度と量でそのようなドメインを豊かにするために、効率的なプロトコルの不足によって妨げられてきた。取クロマチン免疫沈降は、その機能、hPTMs、変異体および共同関連する非histonicタンパク質の面で明確なクロマチン領域を分離するために使用され、それによって我々は、ここにChroP（CHRO MATIN のP roteomics）という名前の新 ​​たに設計されたクロマチンプロテオミクス戦略を、している記述MSにより分析した。我々は彼を説明ヒストンH3上のリシン9のトリメチル化の存在によってマークされた転写サイレントヘテロクロマチン領域に濃縮および分析のためのChroPのの設定、再。達成された結果は、徹底的にヘテロクロマチンプロテオームを特徴付けるChroPの可能性を実証し、クロマチンの異なるタンパク質決定因子が相互作用し、遺伝子座特異的構造的および機能的な構成を確立するために、相乗作用方法を理解するための強力な分析戦略としてそれを証明する。

クロマチン免疫沈降は、ゲノムに沿って、タンパク質やヒストン修飾の局在をプロファイルするために使用される強力な手法です。プロテオミクスと同等で、チップは、目的の変更またはタンパク質と一緒に免疫沈降「餌」として使用される質的にも量的にhPTMs、ヒストン変異体およびクロマチン結合タンパク質を同定するために、MSベースのプロテオミクスが続いている。 図1A?...

Watch this Scientific Journal Video about The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin at JoVE.com

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

ネイティブ組み合わせると高解像度質量分析とクロマチン免疫沈降を架橋することにより、ChroPアプローチはヒストン修飾、変異体及び機能的に異なるクロマチンドメインで相乗非histonicタンパク質の複合プロテオミクスアーキテクチャを分析することができます。

ChroPアプローチは、クロマチンの遺伝子座特異的プロテオームの風景を分析するために、チップと質量分析を組み合わせ

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

the-chrop-approach-combines-chip-mass-spectrometry-to-dissect-locus

Research

JoVE Journal

Biology

18.1K ビュー.  European Institute of Oncology. ネイティブ組み合わせると高解像度質量分析とクロマチン免疫沈降を架橋することにより、ChroPアプローチはヒストン修飾、変異体及び機能的に異なるクロマチンドメインで相乗非histonicタンパク質の複合プロテオミクスアーキテクチャを分析することができます。 

ビデオ: The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDIサンプル調製：超薄層法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

生物学

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

タンパク質の電気泳動分離

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

エレクトロスプレーイオン化質量分析法を用いて酵母Lipidomeの定量評価

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

ヒト細胞において活性化遺伝子発現のアッセイのダイナミックヒストン修飾のクロマチン免疫沈降（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

を使用してクロマチン免疫沈降（ChIP）ショウジョウバエ組織

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

同位体タグ質量分析法を用いたチオールレドックスプロテオームのプロファイリング

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

に基づいた多タンパク質複合体の比較分析のための新たなアプローチ<sup&gt; 15</sup&gt; N代謝標識および定量的質量分析

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕獲化合物質量分析 - 小説のc-ジ-GMPエフェクタータンパク質を同定するための強力なツール

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

レトロウイルス統合サイトの増幅、次世代シーケンシング、およびゲノムDNAのマッピング

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

タンデムアフィニティー精製を使用してのMyoDインタラクトームの同定は、質量分析に結合します

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...