この作品は、ヒューマンフロンティアサイエンスプログラム推進機構（HFSPアワード2007、RGP0058/2007-C）によってサポートされていました。我々はプロジェクトを開始するためと有意義な議論のための教授リチャードロバーツに感謝。

<ol>
	<li>Katayama, H., Oda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+proteomics+for+drug+discovery+based+on+compound-immobilized+affinity+chromatography.">Chemical proteomics for drug discovery based on compound-immobilized affinity chromatography.</a> J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 855, 21-27 (2007).</li><li>Barglow, K. T., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+protein+profiling+for+the+functional+annotation+of+enzymes.">Activity-based protein profiling for the functional annotation of enzymes.</a> Nat. Methods. 4, 822-827 (2007).</li><li>Koster, H., Little, D. P., Luan, P., Muller, R., Siddiqi, S. M., Marappan, S., Yip, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture+compound+mass+spectrometry:+a+technology+for+the+investigation+of+small+molecule+protein+interactions.">Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions.</a> Assay Drug Dev. Technol. 5, 381-390 (2007).</li><li>Dalhoff, C., Hueben, M., Lenz, T., Poot, P., Nordhoff, E., Koster, H., Weinhold, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+S-Adenosyl-L-homocysteine+Capture+Compounds+for+Selective+Photoinduced+Isolation+of+Methyltransferases.">Synthesis of S-Adenosyl-L-homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases.</a> ChemBioChem. 11, 256-265 (2010).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine.">S-Adenosylmethionine.</a> Int. J. Biochem. Cell. Biol. 32, 391-3952 (2000).</li><li>Cantoni, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+methylation:+selected+aspects.">Biological methylation: selected aspects.</a> Annu. Rev. Biochem. 44, 435-451 (1975).</li><li>Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Watson+and+Crick:+DNA+methylation+and+molecular+enzymology+of+DNA+methyltransferases.">Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases.</a> ChemBioChem. 3, 274-293 (2002).</li><li>Chow, C. S., Lamichhane, T. N., Mahto, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+nucleotide+repertoire+of+the+ribosome+with+post-transcriptional+modifications.">Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications.</a> ACS Chem. Biol. 2, 610-619 (2007).</li><li>Grillo, M. A., Colombatto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine+and+protein+methylation.">S-Adenosylmethionine and protein methylation.</a> Amino Acids. 28, 357-362 (2005).</li><li>Fujioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+small+molecule+methyltransferases:+their+structural+and+functional+features.">Mammalian small molecule methyltransferases: their structural and functional features.</a> Int. J. Biochem. 24, 1917-1924 (1992).</li><li>Borchardt, R. T., Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. G., Schlenk, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biochemistry of S-Adenosylmethionine. , 151-171 (1977).</li><li>Hoffman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+the+chiral+and+covalent+instability+of+S-adenosyl-l-methionine.">Chromatographic analysis of the chiral and covalent instability of S-adenosyl-l-methionine.</a> Biochemistry. 25, 4444-4449 (1986).</li><li>Keller, A., Nesvizhskii, A. I., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirical+statistical+model+to+estimate+the+accuracy+of+peptide+identifications+made+by+MS/MS+and+database+search.">Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.</a> Anal. Chem. 74, 5383-5392 (2002).</li><li>Nesvizhskii, A. I., Keller, A., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statistical+model+for+identifying+proteins+by+tandem+mass+spectrometry.">A statistical model for identifying proteins by tandem mass spectrometry.</a> Anal. Chem. 75, 4646-4658 (2003).</li></ol>

ThomasLenz、OliviaGrabner、MirkoGlinski、MathiasDreger、そしてこの記事で使用する試薬の一部を生成するcaprotec bioanalytics社、株式会社に雇用さHubertKosterare。

記述されたプロトコルを以下のときは、次の注意事項とコメントは役に立つかもしれません：これは、その後の厳しい洗浄条件を許可するようにCCの）主要な利点は、CCとMTase間の共有結合の形成にある。共有結合架橋は、UV光（310 nmの最大）によって引き起こされる光反応によって達成される。通常のオーバーヘッドの光は紫外線のごく一部が含まれている、しかし、上caproBoxの制御と冷却照射のオーバーヘッド、あるいは太陽の光に長く曝露によるSAH - CCを保護する。 B）MTasesが分離される元となる生体試料が変性を起こしやすいタンパク質を含む可能性がある、従ってそれは、試料を低温に保つため、常に泡を避けるために必須です。 C）caproBoxが0〜4にサンプルを冷却° C、UV光を発するランプは、しかし、また熱を発する。したがって、それは一時的に照射する前にバイアルを遠心分離する必要があるので、キャップやバイアルの壁に付着したタンパク質は、降水量の種子を形成することはできません。 D）磁性ビーズの再懸濁が（洗浄溶液などにおいて）手で不可能な場合は、すぐに超音波浴中に試料を配置することにより、超音波を適用する。 e）の新鮮な0.2パーセントTFA/60％のACN溶液を調製。我々は、そうでなければ捕捉されたタンパク質がビーズから切断されていない可能性がありますことを発見。 f）に捕捉されたタンパク質の最終的な分析は、LC-MS/MSにより実施される。質量分析法は高​​感度な方法です。それは最後のステップ（ステップ2.11とさらに）で排他的にLC - MSグレード試薬を使用する必要があります。外部のタンパク質源、ほこりからまたは実験者からの発信などのケラチンでの実験のコンタミネーションを避ける。特に最終的な消化のステップの間に、それは、清潔な作業スペースに注意を払うために手袋を着用すると白衣と多分ヘアネットまたは理想的にはクリーンベンチの下に最終的な手順を実行することをお勧めします。フィルタ、分注し、-20℃で保存し、汚染を避けるために一度だけ各アリコートを使用して0.22μmのフィルター、LC MSグレードの水の中でトリプシン消化物に使用する50mMの重炭酸アンモニウムバッファを準備します。トリプシン溶液（クエンシンググレード、ロシュ、凍結乾燥したトリプシンに1mMのHClを添加、0.5μg/μLの溶液を調製）を、4℃で数週間保存することができます。 g）を信頼性の高いマススペクトルを得るためには、ESI-MS/MS分析で安定したスプレーを持つことが不可欠です。 H）本研究で使用したもの以外のLC-MS/MSシステムでは、測定パラメータとペプチド同定アルゴリズムを個別に調整する必要があります。 次の変更を説明するプロトコルに関して可能です：60％ACN/0.2％TFA（ステップ2.11）でビーズからタンパク質を放出する代わりに）、タンパク質は直接trypticallyビーズ懸濁液（同じボリュームなどの内で消化することができます。ステップ5.1）でまたは、SDS - PAGEのために、タンパク質を95ステップ2.9の後に収集されたビーズを懸濁し、加熱することによって解除することができます℃のSDSサンプルバッファーで10分（両方とも、全体の停止または上清のみをロードすることができるためゲルのポケットに）。我々は、ビーズがゆっくりであっても、いくつかの水系洗浄ステップの後で、ビーズトリプシン消化物中に水性のトリプシン消化物の溶液中にポリマーの少量を解放することを発見。ポリマーの汚染は、MSペプチドの同定と干渉し、80％ACNを（少なくとも3回）を使用してビーズから洗浄することができます。 80％ACNの洗浄ステップの後、ビーズを前に、オンビーズトリプシン消化物に水で1回洗浄してください。 B）ストレプトアビジン- horseraddishペルオキシダーゼおよびECL基質を用いたウェスタンブロットはまた、タンパク質にビオチンを含むCCの成功架橋を可視化するために使用することができます。したがって、ステップ3.2の後に得られるいずれのゲルをブロットすることができるまたは、感度が10倍程度であるため、ステップ2.7の後に10μlのサンプルを、ゲルの銀染色よりも高いにもウェスタンブロットで分析することができます。後者の場合にendogeneouslyビオチン化タンパク質はまた、人為的にSAH - CCによるビオチン化タンパク質のほかに検出されることに留意。 c）我々は、架橋反応が溶液4で自由CCとタンパク質の間で行われる&quot;オフビード&quot;の構成、どうか、（ライセート、標的タンパク質、選択性と彼CCの反応性の機能を対処）それは特殊なシステムに依存することを見出したまたは現在説明&quot;上のビーズ&quot;の構成（図1B）は、パフォーマンスが向上します。 一般的には、この方法はまた、あらゆる最先端の安定同位体の蛋白質またはペプチドの標識技術、または2次元ゲル電気泳動によるキャプチャのサンプルの評価と互換性があります。 &quot;オフ - ビーズ&quot;構成では、全細胞（未発表結果）内のタンパク質をキャプチャすることも可能です。さらに、薬剤またはタンパク質の補因子結合部位は、MSペプチド配列決定によりタンパク質配列内にCCのすぐ近くの架橋の位置を決定することにより説明することができます。蛋白質への小分子の結合様式は異なるC言語を使用して調べることができます選択性の機能と異なるリンカー長でhemical取り付け位置。として選択する機能によってアドレスのタンパク質のタンパク質結合パートナー（PRMAの基質としてRplK）また、本研究で示されているか、または未知の小分子のタンパク質の相互作用（GlnAにSAH）を識別することができます。要約、CCの追加機能、光架橋反応は、高感度で、複雑なタンパク質混合物から低濃度タンパク質や機能性タンパク質のファミリーの分離と同定を可能にし、低分子化合物を研究するための追加のツールを科学者を提供 - タンパク質相互作用。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SAH caproKit&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-1010-050 (50 reactions) 1-1010-010 (10 reactions)</td>
<td>Includes the SAH-CC, SAH competitor, streptavidin coated magnetic beads, capture buffer, and wash buffer</td>
</tr>
<tr>
<td>caproBox&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5010-003 (110 V) 1-5010-004 (230 V)</td>
<td>For reproducible photo-activation while cooling the samples</td>
</tr>
<tr>
<td>caproMag&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5100-001</td>
<td>For easy handling of magnetic particles without pipetting</td>
</tr>		</tbody>
		</table>
		</div>

profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)

アフィニティークロマトグラフィーなど1または活動ベースのタンパク質プロファイリング2のような機能性小分子とタンパク質の相互作用に基づいてプロテオームの複雑さを軽減するためのさまざまなアプローチがあります。三官能キャプチャ化合物（CCS、図1A）3は 、一般的なアプローチの基礎とされている初期の平衡駆動型の低分子プローブ（ここで選択性の機能、S -アデノシル- L -ホモシステイン、SAH、図1A）との間の相互作用と標的タンパク質は、不可逆的にCCの独立した光活性化反応の機能（ここでphenylazide）と標的蛋白質の表面との間の光架橋により固定されています。固相（ここでストレプトアビジン磁気ビーズ）の助けを借りて、複雑な生物学的な混合物からタンパク質複合体 - ソート関数（ここでビオチン）は、CCを分離するのに役立つ。実験の二つの構成が可能です：&quot;オフビーズ&quot;4または現在説明&quot;上のビーズ&quot;の構成（図1B）。選択性の機能は、関心のあるほぼすべての小分子（基質、阻害剤、薬物分子）があります。 （SAM、図1A）S -アデノシル- L -メチオニンは、おそらくATP、自然5、6で最も広く使用されている因子に2番目です。それはDNA 7メチル化SAM依存メチルトランスフェラーゼ（MTases）、、RNA 8、蛋白質9、または小分子10で触媒される化学反応を持つすべての生物の主要なメチル基供与体として使用されます。多様な生理学的なシナリオでのメチル化反応の重要な役割（遺伝子制御、エピジェネティクス、代謝）を考えると、MTasesのプロファイリングは、キナーゼのプロファイリングなど、機能的プロテオミクスにおける同様の重要性になることを期待することができます。彼らのプロファイリングのための分析ツールは、しかし、利用されていない。我々は最近、この技術格差（図1A）を埋めるために選択性のグループとしてSAHとCCを導入しました。 SAH、メチル転移後のSAMの製品は、既知の一般的なMTase製品の阻害剤11です。このような理由から、天然補酵素SAMが原因で、その化学的不安定性の12因子の他の部分を転送するかだけでなく、ラジカル反応を開始、さらに酵素で使用されているため、SAHは、ターゲットMTasesへのCCの理想的な選択性の関数です。ここで、我々は、 大腸菌の株DH5α（ 大腸菌 ）からMTasesと他のSAH結合タンパク質をプロファイリングすることによって好ましいモデルを務めている最高の特徴と原核生物、のいずれかをSAH - CCとCCMSのユーティリティを報告する数え切れないほどの、生化学、生物学、およびバイオテクノロジーの研究で生物。写真活性化架橋は、実験の収量と感度を向上させ、特異性は容易に自由SAHの過剰を使用して競合実験のためにテストすることができます。

Watch this Scientific Journal Video about Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS at JoVE.com

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

キャプチャ化合物は、光架橋および精製に続いて​​機能的な可逆小分子 - タンパク質相互作用により、プロテオームの複雑さを軽減するために三官能性低分子化合物です。ここでは、使用してキャプチャーコンパウンドを使用<em&gt; S</em&gt; -アデノシル-<sub&gt; L</sub&gt; -ホモシステイン結合選択性の関数としてのメチルトランスフェラーゼを分離する<em&gt;大腸菌</em&gt;全細胞は、溶解液とMSでそれらを識別する。

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 ...

profiling-methyltransferases-other-s-adenosyl-l-homocysteine-binding

Research

JoVE Journal

Biology

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

15.5K ビュー.  caprotec bioanalytics GmbH. キャプチャ化合物は、光架橋および精製に続いて​​機能的な可逆小分子 - タンパク質相互作用により、プロテオームの複雑さを軽減するために三官能性低分子化合物です。ここでは、使用してキャプチャーコンパウンドを使用 S -アデノシル- L -ホモシステイン結合選択性の関数としてのメチルトランスフェラーゼを分離する

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

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 ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

構造質量分析法によって大きなタンパク質複合体の分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

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 ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

LAESI質量分析による生体組織とバイオフィルムの大気圧分子イメージング

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

大気圧質量分析法による単一細胞の直接分析

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

質量分析法とあいまってペプチドイムノエンリッチメントを使用したタンパク質の定量化

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

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 ...

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 ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

メチルトランスフェラーゼと補因子類似体核酸およびタンパク質の配列特異的標識化

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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 -ホモシステイン結合タンパク質

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 -ホモシステイン結合タンパク質}