この作品は、エネルギーバイオサイエンス研究所の助成金OO0G01によって賄われていた。

オリンプメソッドはglycosylhydrolaseとしてエンドグルカナーゼを用いて、双子葉植物、キシログルカンの細胞壁に存在する主要なヘミセルロースに例示される。方法は、植物組織の源としての全体シロイヌナズナ実生を用いて実証されています。酵素と細胞外マトリックス材料は、同じ手順を用いて所望の分析に応じて置換することができます。 1。細胞壁の分離<ol><li>収穫5人が、5日齢全体シロイヌナズナの苗や、ご希望の材料の相当額と1.5mlの反応管に移す。材料は、チューブの底部に配置されていることを確認します。 </li><li>液体窒素でのサンプルとスナップ凍結の上に反応管に2つ3ミリメートルの金属のボールを追加。 </li><li>ボールミル（2:30分、25Hzの）を使用して凍結サンプルを挽く。 </li><li>サンプルに1mLの70％水性エタノールを加えます。磁石を用いて金属球を取り外します。徹底的にボルテックス。 </li><li>ペレットに10分細胞壁物質を含有するアルコール不溶性残渣を14,000 rpmでサンプルを遠心分離します。 </li><li>慎重に吸引して上清を取り除く。ペレットを妨害しないことを確認してください。 </li><li>クロロホルム/メタノール（1:1 v / v）溶液1mLの追加。ペレットを再懸濁させる徹底的にボルテックス。 </li><li> 10分間14,000 rpmでサンプルを遠心し、慎重に吸引して上清を取り除く。ペレットを妨害しないことを確認してください。 </li><li>コンセントレータを使用してサンプルを乾燥させる。 </li></ol> 2。オリゴ糖の可溶化<ol><li>可溶化のために、そのオリゴ糖の形で特定の多糖類は、適切なバッファーの25μlのに乾燥したペレットを再懸濁します。エンドグルカナーゼ50mmのギ酸アンモニウム、pH4.5の場合は、使用されています。エンドグルカナーゼの0.2Uを追加。ダウン渦サスペンションとスピン液体。 </li><li> 37℃で16時間サンプルをインキュベート° Cのインキュベーターで、300rpmで振盪。 </li><li>コンセントレータを使用してダイジェスト（水）の溶剤を削除します。 </li></ol> 3。放出オリゴ糖のMALDI - TOF分析<ol><li> BioRex MSZ 501陽イオン交換樹脂ビーズは、消化サンプルから塩と酵素を除去するために使用されています。空のカラムに一定量を配置し、多量の水でビーズを洗浄することにより条件ビーズを。 </li><li>消化し、乾燥サンプルに5〜10 BioRexビーズを追加。その後、水の材料の5μlの少量を使用することができるために、10μlの水を加えます。簡単にチューブを回転させて、溶液中のビーズを浸す。室温で少なくとも10分間インキュベートする。 </li><li> MALDIターゲットプレートにスポット2μlのマトリックス（2,5 - ジヒドロキシ安息香酸（DHB）、水で10mg/ml）。均一なマトリックスの化学物質の結晶につながる真空下で溶媒マトリックスを蒸発させる。 </li><li>ターゲットプレート上にマトリックスの結晶の上に脱塩試料溶液のスポット2μlの。 あなたが多数のサンプルを発見する場合のみ3分の最高のためにスポッティング続ける。この時間枠は、最初に斑点を付けられたサンプルがまだ乾燥していないことを保証します。最後の斑点のあるサンプルに再溶解し、マトリックスとサンプル糖鎖分子の完全な混合を確実にするために追加の2分間待ちます。その後、真空下でターゲットプレートを乾燥させる。 サンプル/マトリックス混合は、均質な結晶化を有効にするために1分も経たないうちに結晶化する必要があります。 </li><li> MALDI - TOF質量分析計への場所のターゲットプレート。マシンは、20000Vと350nmの遅延の加速電圧と正のリフレクトロンモードに設定する必要があり、選択されている質量範囲500から3000ダ（予想されるオリゴマーに依存して変更される可能性がある）はずです。レーザーを発射起動し、代表的な平均のスペクトル（図2A）を生成するためにコンパイルされているサンプル当たり100から200のスペクトルを、収集する。特定のオリゴ糖を表すイオンが電荷（m / z）比を、その質量によって識別することができます。関心のあるすべてのイオンのイオン強度（ピーク高さ）サンプル中の各オリゴ糖の相対量（図2B）、結果として100％まで追加する必要があります。 </li></ol> （4） その場オリンプの分析では オリンプは、任意の壁の準備手順を省略する組織に直接使用することができます。例として、植物組織の源としてシロイヌナズナの黄化胚軸が使用されます。再び、エンドグルカナーゼは、ヘミセルロースのキシログルカンの構造を決定するために使用されます。酵素と組織の材料は、同じ手順を用いて所望の分析に応じて置換することができます。 <ol><li>黄化苗を収穫し、空のMALDIターゲットプレート上に置き、苗を乾燥させて。 </li><li>所望の位置に乾燥させた苗を0.5μlエンドグルカナーゼを（50mmのギ酸アンモニウムに溶解0.4U/μl、、pH4.5）を追加します。酵素の低下が部分的にターゲットプレートに触れていることを確認してください。 </li><li>その酵素ドロップが乾燥を避けるために、飽和湿度で密閉容器にMALDIターゲットプレートを置きます。 1のチャンバー内にプレートをインキュベート37 6H℃、 </li><li>植物組織および真空下で酵素のドロップとMALDIターゲットプレートを乾燥させます。 </li><li>各乾燥酵素のスポットの上に、0.5μlの行列を（10mgの/ L DHB）を追加します。それが2分間放置する。 </li><li>真空下MALDIターゲットプレートを乾燥させます。 </li><li> MALDI - TOFのサンプルを分析する。 MALDI - TOF質量分析計への組織および酵素/マトリックススポット（複数可）でターゲット板を配置。マシンは、20000Vと350nmの遅延の加速電圧と正のリフレクトロンモードに設定する必要があり、選択されている質量範囲500から3000ダ（予想されるオリゴマーに依存して変更される可能性がある）はずです。 </li><li>組織へNEXTマトリックス/試料結​​晶にレーザーを発射開始。あなたのような分子のフライトの時はoffとなるような場合には、組織自体に該当していないことを確認してください。代表的な平均のスペクトル（図3）を生成するためにコンパイルされているスポット、当たり20から50前後のスペクトルを収集する。特定のオリゴ糖を表すイオンが電荷（m / z）比を、その質量によって識別することができます。イオン強度（イオンの高さ）が報告することができ、関心のすべてのイオンは試料中の各オリゴ糖の相対量で、その結果100％まで追加する必要があります。 </li></ol> 5。代表的な結果 シロイヌナズナ実生におけるヘミセルロースのキシログルカンの存在のオリンプ分析の例を図2に示されています。イオンの質量差と知られて十分に特徴付けられた酵素（エンドグルカナーゼ）特異性に起因するイオンは、特定のオリゴ糖の構造（図2A）に割り当てることができます。明らかに、構造異性体を区​​別することができます。様々なオリゴ糖の相対量の決定のための基本的な前提は、（図2B）、その質量分析の応答係数は、それらのオリゴ糖のために非常に似ていることです。ここに示すように、オリンプ定量化は非常に再現性です。しかし、メソッドの堅牢性は、様々なオリゴ糖のイオン対雑音比信号に大きく依存していることに注意してください。例えば塩またはオリゴ糖のより少量の混入は、その比率を減らすことができます。 組織自体のオリンプ分析は、（その場分析で）非常に小さいと定義されている領域の研究を可能にし、非常に少ないサンプル調製を含む。例では、定性的（同じイオン）（図3）ここで紹介しませんが、定量的な違いは、（イオン強度のばらつき） シロイヌナズナ実生の茎と根組織の間で観察された。オリンプ法の順列は、このように組織、&quot;イメージング&quot;につながる可能性があります。 <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="図1" /> 図1植物源として全体シロイヌナズナの苗を使用してオリンプ手順のフローチャート。最初に、組織が ​​漬けられ、細胞壁の材料は（写真は藤野らから変更。10）用意されています。その後、特定の壁の多糖類のオリゴ糖は、特定の加水分解酵素を使用して解放されています。最後に、水溶性オリゴ糖の相対的存在量をMALDI - TOF質量分析法を用いて決定されます。 <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="図2" /> 図2。オリンプにより決定されたシロイヌナズナから黄化実生におけるキシログルカンのオリゴ糖の相対的な量。 A）代表キシログルカンオリゴ糖質量スペクトルを、それぞれのイオンは、特定の糖鎖構造を表し、構造異性体を区​​別することができます。相対的なオリゴ糖豊富な測定のためのB）対応するバーの図。 <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="図3" /> 図3。例として、 シロイヌナズナの黄化実生を用いてその場オリンプの分析では 。それぞれの酵素/消化液滴（色付きの丸）が独立して分析することができ、対応するマススペクトルが得られ、分析することができます。 <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="図4" /> キシラナーゼ（Megazyme）とススキの葉の材料を消化し ​​て得られたヘミセルロースのキシランの図4。オリンプスペクトル。

<ol>
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利害の衝突は宣言されません。

ここで紹介するオリンプ方法は、細胞外マトリックスに存在するポリマーの非常に敏感かつ迅速な分析が可能になります。オリンプは、後続のMALDI - TOF分析とオリゴマーの酵素的放出を兼ね備えています。 MALDI - TOFスペクトルの生成は、1分未満を受け取り、それ故にオリンプは、このような変異体の画面のようなハイスループット試験を含む広範囲のアプリケーションに適しています。オリンプは、植物の多糖類に限定されるものではなく、潜在的にのみ特異的加水分解酵素の利用可能性によって制限ポリマー、の広い範囲に適用することができます。しかし、オリンプの制限は、ポリマーの絶対的な豊かさが得られないということです。 オリンプが種の多様性の細胞外マトリックスに存在する多糖類の様々の構造を研究するために使用する前に、前述のとおり。例として、図4は、草種の主要なヘミセルロース、キシランのオリンプスペクトルを表しています。ここでは、温帯草ススキ由来の細胞壁の材料は、キシラナーゼを用いて消化した。

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

oligo mass profiling (olimp) of extracellular polysaccharides

環境への細胞の直接接触は、細胞外マトリックスによって多くの生物に仲介されている。細胞外マトリックスの一つの一般的な側面は、彼らは複雑な糖の糖タンパク質の形で部分、プロテオグリカン、および/または多糖類を含んでいるということです。例としては、繊維状のタンパク質およびプロテオグリカンや植物と菌類の多糖類ベースの細胞壁を主とする人間と動物細胞の細胞外マトリックス、プロテオグリカン/細菌の糖脂質ベースの細胞壁が含まれています。すべてのこれらのglycostructuresは、細胞間および細胞 - 環境コミュニケーションとシグナル伝達において重要な役割を果たしている。 細胞外マトリックスの異常な複雑な例では、高等植物の細胞壁に存在しています。その壁は、最大75％乾燥重量に、糖からほとんど完全に成って、そしてこの地球上に存在する最も豊富に存在する生体高分子から構成されています。したがって、研究は化石燃料から得られる石油化学製品を置き換えるために、カーボンニュートラルな再生可能資源として最適なこれらの材料を活用する方法を実施しています。燃料転換のための主な課題は、このユニークなbiocompositeに存在するユニークなglycostructuresによる酵素的または化学的劣化に壁の反抗のまま。 ここで、我々は、植物細胞壁のglycostructuresの迅速かつ高感度分析法を提示する。このメソッドオリゴマスプロファイリング（オリンプは）特定のglycosylhydrolasesとマトリックス支援レーザー脱離/イオン化飛行時間型質量分析（MALDI-TOF/MSを用いて可溶化したオリゴ糖混合物のその後の分析を容易にする壁材料からのオリゴ糖の酵素のリリースをベースにしています1）（図1）。オリンプは、完全な分析のためにのみ5000細胞の壁は、組織自体が2で実行できる必要があり、ハイスループット分析3に従順です。可溶化されたオリゴ糖の絶対量がオリンプによって決定することはできませんが、様々なオリゴ糖のイオンの相対量は、壁に存在する天然多糖の置換パターンについての洞察を与えるマススペクトルから線引きすることができます。 オリンプは、特定の酵素4の可用性によって制限壁ポリマー、多種多様な分析をするために使用することができます。例えば、植物細胞壁の酵素に存在するポリマーの分析のためにエンド -β-（1-4）-キシラナーゼ6,7を使用してキシログルカナーゼ5、11、12、13、キシランを用いたヘミセルロースのキシログルカンを分析するために用意されています、またはポリガラクツロナーゼおよびメチルエステラーゼ8の組み合わせを使用して、ペクチン多糖類のため。さらに、オリンプglycosylhydrolaseとさえ糖転移酵素活性のと同じ原理を使用しては監視し、9を決定することができます。

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

迅速な方法は、細胞外マトリックス中の多糖類の構造についての洞察を得るために説明されています。方法はglycosylhydrolasesの特異性と材料の微量を分析できるように質量分析の感度を利用しています。この手法は、組織自体に直接使用するための適応です。

細胞外多糖のオリゴマスプロファイリング（オリンプ）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

oligo-mass-profiling-olimp-of-extracellular-polysaccharides

Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K ビュー.  University of California, Berkeley. 迅速な方法は、細胞外マトリックス中の多糖類の構造についての洞察を得るために説明されています。方法はglycosylhydrolasesの特異性と材料の微量を分析できるように質量分析の感度を利用しています。この手法は、組織自体に直接使用するための適応です。

ビデオ: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

ルシフェラーゼ免疫系による抗体のプロファイリング（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル- L</subキャプチャの複合質量分析法（CCMS）別> -ホモシステイン結合タンパク質

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成体マウスの心筋細胞の単一細胞転写プロファイリング

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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 ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

マイクロアレイを用いた植物細胞壁ポリマーの糖鎖プロファイリング

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

質量分析のためのペプチドに組織および消化から細胞外マトリックスタンパク質の濃縮

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

工場ポリソームプロファイリングのための簡単​​な方法

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

ハイコンテンツ顕微鏡を用いた細胞レドックスプロファイリング

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...