この作品は、衛生費GM - 26738（JATまで）の国立研究所からの一部でサポートされています。

1。 PAK2活性化<ol><li>バッファの12 mg / mlのPAK2の20μlにカスパーゼ3の適切な事前検証済みの金額を追加（pH7.5の50mMトリス- HCl、150mMのNaCl、2 mMのDTT）、34℃でインキュベート℃で30分間完全に切断するとアクティブにPAK2へ。 </li><li> SDS - PAGE（10％）とCoommassieブルー染色により切断産物を分析する。 </li><li> 10 mg / mlのPAK2の最終濃度20μlに、pH7.5の活性化緩衝液B（50mMトリス- HCl、150mMのNaCl、15 mMのMgCl 2、10mMのATP）に切断されたPAK2を追加し、34でインキュベート℃で30分間完全に8サイトで自己リン酸化によってPAK2をアクティブにする。 </li><li>としてSDS - PAGE 10で決定したP34とP27の断片の移行により、PAK2の完全切断を確認してください。自己リン酸化状態の適切な検証は、リン酸化ペプチドを検出するために（32 P）ATPまたは質量分析計を用いた別の実験でオートラジオグラフィーを介して行われる。 </li></ol> 2。識別ペプシン消化PAK2フラグメントの<ol><li>使用するために二回前にアガロースビーズを洗浄するためにpH 2.5の0.1％トリフルオロ酢酸（TFA）を1 mlを追加。その後、ビーズを除去するために2000 × gで15秒間のサンプルを遠心してください。 </li><li> 0.1％TFA 100μlにPAK2（150mMのNaCl2μlの20μgの、pH7.5の50mMのTris - HCl、2 mMのDTT）を追加し、ペプシン - 共役アガロースビーズを30μlと5分間サンプルをインキュベート。 </li><li> pH2.5で0.1％TFAの主溶媒系逆相HPLC C18カラムを平衡化する。 0.2ml /分の流速で0.1％TFAを含む80％アセトニトリル - ペプシン消化物（50μl）を0のリニア、100分の勾配を用いて溶出される。画分を毎2分を集めている。 </li><li> （3時間）速度- VACでHPLC画分を乾燥し、アセトニトリル（5μL）とpH2.5の0.1％TFA（5μl）の溶液中で試料を懸濁します。 </li><li>当初は、各フラクションをMALDI - TOF PerSeptiveバイオシステムズボイジャーDEのSTR（PE Biを用いてペプチドをスクリーニングされosystems、フォスターシティ、カリフォルニア州、米国）。 </li><li>ペプチドを含んだ、16画分はフラグメントを識別するために、Q - TOF質量分析計とQSTAR XL oMALDI MS / MSを用いたタンデム質量分析に供される。 </li><li>各ペプチドの配列は、プロテインプロスペクターのウェブサイト（使用PAK2の一次配列に基づいて理論的なm / zの比を有するタンデム質量分析によって生成された生成物イオンによって検証されます<a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ）11。 </li></ol> 3。アミドH / D交換の測定<ol><li>水浴で25℃、H / D交換のために必要なすべてのソリューションと試薬° Cを保持する。 ATPを水に溶解し、pHは、使用前に7.0に調整されます。 D 2 Oバッファの追加4mmのATPは、希釈後PAK2から解離ATPを防ぐためです。 </li><li> （20μgの2μlのにバッファリングされたD 2 O（50 mMのMOPSのpHは6.98、125 mMのNaCl）を18μlを添加することにより、H / D交換を開始）0時10 mg / mlのおよび7.0のpHのPAK2レベルで、その結果150mMのNaCl、50mMのTris - HCl（pH7.5）及び2mMのDTTを含む緩衝液中PAK2、℃の</li><li> 0、0.5、1、3、5、および10分、または24時間H / D交換の三連でサンプルをインキュベートする</li><li> 200μlの最終容量で2.5にpHをもたらすpHを2.2、氷冷0.1％TFA 180μlを添加して、H / D交換のプロセスを急冷。 ATP、pHは2.2で氷冷0.11％TFA 180μlを含むサンプルの場合は焼入れ状態で2.5のpHを維持するために使用されます。 </li><li>活性ペプシン-共役アガロースビーズを30μlに各急冷試料のアリコート（100μlの）を追加します。ペプシン消化は5分間氷上に進行させると、サンプルは30秒ごとにボルテックスしている。 </li><li> 4℃で5000 × gで15秒間のサンプルの遠心分離により消化を終了° Cは、ペプシンを削除する。 </li><li>急速に3回以上の-80℃で液体N 2と店舗でサンプルを凍結するMALDI - TOF分析に先立って日。 </li><li> MALDIのマトリックスは、5 mg / mlのα-シアノ-4 - ヒドロキシアセトニトリル、エタノール及びpH 2.5の0.1％TFA溶液（午前1時01分01秒）に溶解し、0℃で開催された酸（CHCA）です。 </li><li>部分的に迅速に各サンプルを解凍し、4℃MALDIのターゲットプレート上にマトリックスとスポットの1μlを各サンプルを1μlを混合。 </li><li>前のMALDI - TOF分析に30秒のスポットを乾燥させるプレートに適度な真空を適用します。サンプルの融解とスペクトルの取得との間の時間は3分程度です。 </li><li> PerSeptiveバイオシステムズボイジャーDEのSTRとMALDI - TOF質量スペクトルを取得する。 20,000 Vの加速電圧、および180 nsのパルスの遅延と遅延抽出を使用して65％グリッド電圧で、10万のデータチャンネルで、2 GHzのサンプリングレートでデータを取得する。百スキャンは2分で平均化されています。 MALDI - TOF装置はあらゆるブランドや特定のソフトウェアに限定されるものではありません。 </li><li> calculにデータExplorer 4.0のコンピュータプログラムを使用して、各フラグメントの重水素化を食べた。 </li><li> N -末端アミド（ 図1）を除き、バックボーンのアミド類である可能性のあるすべての交換可能なサイト、で割ったH / D交換の24時間での実際の組み込まれた重陽子の数の比に基づいて、バックの交換を計算する。 </li></ol> 4。代表的な結果： カスパーゼ切断と自己リン酸化は、SDS - PAGE Commassie染色によって検証されます。非アクティブなPAK2は58 kDaの単一バンドです。 PAK2のカスパーゼ切断が完了し、別々に移行する二つの断片、P27とP34を生成します。自己リン酸化p27のとP34を示し、完全にリン酸化されたp27の断片（ 図2）の遅滞マイグレーションによるゲル上でのオーバーラップの移行。 H / D交換実験は、0〜10分（ 図3）の間に複数回行われている。例として、M / ZPに重水素結合の時間経過時間をかけて大量の封筒のシフトで示されるように非アクティブPAK2のEAK 1697.85は、複数の同位体のピークで構成されています。 <img alt="図1" src="/files/ftp_upload/3602/3602fig1.jpg" /> 図1重水素化の計算のための方程式。 <img alt="図2" src="/files/ftp_upload/3602/3602fig2.jpg" /> 図2自己リン酸化とPAK2のカスパーゼ切断。カスパーゼ切断されたPAK2（左車線）、無傷の非アクティブPAK2（中央車線）とカスパーゼ切断された自己リン酸化PAK2（右レーン）をSDS - PAGEとCoommassieブルー染色により分析した。スー、YHら2から適応。 <img alt="図3" src="/files/ftp_upload/3602/3602fig3.jpg" />非アクティブなPAK2用重水素結合の時間経過の図3。質量スペクトル。非アクティブなPAK2は、H / D exchaに供した00〜10分間のゲイン誤差とMALDI - TOFで分析。 H / D交換の時間経過時のピークのm / z 1697.85のMALDI - TOFの拡張同位体分布の例が示されている。赤い点線は、大量のエンベロープの平均値を示し、大量の封筒のシフトは、ペプチドの重水素化を示しています。

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+accurate+mass+measurement+(+/-+10+ppm)+in+protein+identification+strategies+employing+MS+or+MS/MS+and+database+searching.">Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.</a> Anal. Chem. 71, 2871-2882 (1999).</li></ol>

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ペプシン消化断片の同定は、H / D交換実験の重要なステップです。ペプチドの不正確な同定は、間違った結論を導くことができる。ペプシン消化PAK2は、きれいなバックグラウンドを得るために、逆相HPLC C18カラムに通して溶出させる。我々の実験では、40画分の合計を集め、MALDI - TOFに供した。複数のペプチドは、各画分で同定することができた。ペプチドはその配列を同定するためにタンデム質量分析計（Q - TOF MS / MSとoMALDI MS / MS）を行った。ペプチドのMS / MSスペクトルはペプチドの同定を確認するために少なくとも3つの製品のイオンを持つ必要があります。 データエクスプローラ4.0のコンピュータプログラム（アプライドバイオシステムズ）は、初期のベースライン補正やノイズフィルタを実行します。各スペクトルは2つのシークエンスピークに基づいて校正する必要があります。我々は、923.45と1697.84ですundeuteratedのm / zの理論的質量によって私達のスペクトルを校正。 T彼は、キャリブレーション後の質量精度は10ppmに達することができる。重水素化されると、モノ同位体は、より高い質量にシフトする場合があります。これらのm / zの比にユニタリステップの増加は、重水素化に関連付けられている強化された大衆が得られた。大量の封筒のピーク強度は、重水素化のレベルには影響しません。しかし、特定の質量の封筒の同位体ピークのピーク強度は、平均的な質量の計算のための非常に重要です。取り込まれた重陽子の計算の最初のステップは、重水素化サンプルから非重水素化試料のペプチド質量を引いている。他の3つの要因は、D 2 O希釈、側鎖に残留重陽子とバックの交換は、さらに計算（ 図1）で考慮する必要があります。 D 2 Oの希釈は、H / D交換を開始するサンプルとD 2 Oのバッファを混合後、D 2 Oの希釈率です。ペプシン消化物の側鎖中の残留重陽子（4.5％）EDのペプチドは、減算する必要があります。バックの交換は、質量測定のプロセスのすべての重水素とペプシンで消化されたペプチドの重水素化の必然的な損失です。 H / D交換の24時間後にほとんどの溶媒にアクセス可能なペプチド（m / zの= 1105.60）は、完全な重水素化の領域を表します。ペプチドの各々に対して組み込ま重陽子の数は、重水素化や非重水素化PAK2消化ペプチドの重心の差です。最も高度に重水素化ペプチドのm / z 1105 PAK2は、H / D交換の24時間にいたときには完全な重水素化を表すために選ばれました。戻る交換比率は、ペプチドのm / z 1105のすべての可能な交換可能なサイトで割ったH / D交換の24時間で実際の組み込まれた重陽子の数によって算出した。

<table border="1"></table><table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td> D 2 O </td><td>アルドリッチ</td><td> 7789-20-0 </td><td></td></tr><tr><td> α-シアノ-4 - ヒドロキシ桂皮酸</td><td>シグマ</td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI - TOF </td><td> PEバイオシステムズ</td><td>ボイジャーDEのSTR </td><td></td></tr><tr><td> Q - TOF質量分析計</td><td> Q - TOFアルティマ - グローバル</td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI MS / MS </td><td>アプライドバイオシステムズ</td><td></td><td></td></tr><tr><td>データエクスプローラ4.0のコンピュータプログラム</td><td>アプライドバイオシステムズ</td><td></td><td></td></tR&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

質量分析法と相まってアミド水素/重水素交換（H / D交換）が広くタンパク質 - タンパク質相互作用、タンパク質立体構造の変化、タンパク質のダイナミクスとタンパク質 - リガンド相互作用のインターフェースを分析するために使用されています。バックボーンアミドポジションのH / D交換は、質量分析法1,2,3によってタンパク質のマイクロの領域の重水素化率を測定するために利用されている。この方式の分解能は、通常30から20残基の範囲であるペプチドへの関心の重水素化タンパク質のペプシン消化によって決まります。質量分析法により測定されたH / D交換の決議は、NMRの異種単量子コヒーレンス（HSQC）法で測定した1残基の解像度よりも低いですが、H / D交換の質量分析の測定は、のサイズによって制限されていませんタンパク質4。 H / D交換は、タンパク質の立体構造を維持する水溶液中で実施される。我々は、util、そのメソッドを提供する代わりに、HPLC / ESI（エレクトロスプレーイオン化）- MSシステム5,6の検出2のMALDI - TOFを、izes。 MALDI - TOFは、この場合プロテインキナーゼPAK2（また、γ- PAKと呼ばれる）で、​​消化タンパク質のペプチドの精密質量の強度データを提供します。パック2のタンパク質分解は、オフラインペプシン消化で行われる。ユーザーは、質量分析に接続されているHPLCおよびペプシン列へのアクセス権を持っていない、またはHPLCでのペプシンの列には、例えば、最適な消化のマップでは発生しません場合は、この代替法、、重く、ジスルフィド結合分泌ホスホリパーゼA 2 （SPLA 2）。この方法を利用して、我々は成功したカスパーゼ3切断と自己リン酸化7,8,9によってPAK2の活性化中に重水素化レベルの変化をモニターした。

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI - TOF質量分析法は、正常にプロテインキナーゼPAK2活性化のアミド水素/重水素交換を監視するために利用された。

PAK2活性化のアミド水素/重水素交換とMALDI - TOF質量分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

amide-hydrogendeuterium-exchange-maldi-tof-mass-spectrometry-analysis

Research

JoVE Journal

Biology

17.7K ビュー.  Tunghai University. MALDI - TOF質量分析法は、正常にプロテインキナーゼPAK2活性化のアミド水素/重水素交換を監視するために利用された。

ビデオ: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

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

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 ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T波イオンモビリティー質量分析：タンパク質複合体解析のための基本的な実験手順

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

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 ...

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 ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF質量分析およびカスタムデータベースを使用すると、ユニークな洞窟環境（カーチェナー洞窟、AZ、USA）を常在菌を特徴づけるために

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

絶対タンパク質定量のための質量分析法を選択反応モニタリング

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

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 ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

シングルプローブの応用：周囲条件下での質量分析イメージングと単一細胞解析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

ヒストンの抽出からデータ解析まで：ボトムアップ質量分析法を用いてヒストンの翻訳後修飾の解析のための完全なワークフロー

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...