We thank Alberto Reinders for his work in optimizing the CCMS conditions for P. aeruginosa. We also thank Pablo Manfredifor the annotation of the P. aeruginosa proteins. This work was supported by the Swiss National Science Foundation (SNF) Sinergia grant CRSII3_127433.

1.溶解物の準備<ol><li> P.を育てる希望ODにLBにおける緑膿菌細胞 。 注：ガイダンスについて：対数期の培養のための固定相培養のため≈100ミリリットル文化/サンプルと≈500ミリリットルのcultur /サンプル（OD 600nmが = 0.5）を使用します。 </li><li> 5000のx gで20分間の遠心分離によってペレット。 </li><li>同様に1ミリリットルの溶解緩衝液（6.7 mMのMES、6.7 mMのHEPES、200mMのNaCl、6.7 mMのKAC、DDTを1mM、pH7.5）中、およびプロテアーゼ阻害剤を追加し（EDTAを含まない完全なミニ）でペレットを再懸濁0.5〜1グラムDNアーゼIとして。 </li><li>溶解20,000psiでフレンチ圧力セルを通じて3継代によって細胞、（材料リストを参照してください）​​。 </li><li>超遠心機で4℃で1時間、100,000×gで細胞溶解物。 </li><li>上清を保存します（ステップ2に進み）。 </li><li>ピペッティングにより溶解バッファー1×1ミリリットルとのペレットを洗浄。 </li><li> 4℃で1時間100,000×gで超遠心機。 </li><li>ペレットをフラッシュ凍結膜タンパク質の捕捉に使用するまで-20℃で液体窒素や店舗（ステップ3を参照）。 </li></ol> c-ジ-GMP無料と他のヌクレオチドの2の除去（可溶性画分のみ） <ol><li>冷溶解緩衝液10mlをPD10脱塩カラム（参照物質一覧）を洗浄する。 </li><li>ヌクレオチドを​​除去するために、PD10上に上清（≈1ミリリットル）を注ぐ。 </li><li> 4mlで溶出する冷溶解緩衝液（500μlのステップ）。 </li><li> Bradfordアッセイによって決定されるように最も濃縮画分を選択し、それらをプールする。 </li></ol> 3.ペレットの再懸濁および可溶化（膜画分のみ） <ol><li> （界面活性剤なし）1×捕捉緩衝液の500〜1000μlの中にペレットを再懸濁した（ 表1を参照のこと）。 注：ペレットが再懸濁することは困難である。大体うまく解決策を均質化する注射器27のGを使用するように、その後、ペレットを再懸濁するために、ITをピペットで第一ピペットアップにとダウンをお勧めします。 </li><li> 1％を加える（w / v）のn-ドデシルβ-D-マルトピラノシド（DDM）。 </li><li>回転ホイール上に少なくとも2時間（またはO / N）、4℃でインキュベートする。 </li><li> 4℃で1時間100,000×gで超遠心機。 </li><li>上清を収穫。 </li></ol> 4.タンパク質濃度測定<ol><li> （膜画用のBCAアッセイによる）ブラッドフォードアッセイによりタンパク質濃度を測定します。 </li><li> 10 mg / mlの総タンパク質濃度を設定する。 </li></ol> 5.キャプチャ<ol><li> GDP、GTP、ATP、CTPの1 mMの、そして5倍キャプチャバッファ（100mMのHEPES、250 mMのKAC、50 mMのMgAc、5％グリセロール、pH7.5）を（ 表1）の20μlのタンパク質300μgのを混ぜる。 H 2 Oで100μlに反応容量を調整 注：総ボリュームは（ステップ5.3および表2を参照）競合制御の場合のCDG-CC及びc-ジ-GMPのボリュームが含まれています。 注：全ての実験は、200で行った＃181; L 12-チューブPCRストリップ（サーモサイエンティフィック）。 注：膜画分のために、常に8 M尿素（ステップ7.1）中のタンパク質可溶化工程まで、臨界ミセル濃度（w / vの0.01％）を上回るDDM濃度を維持することを確認してください。 </li><li>回転ホイール上で30分間、4℃でインキュベートする。 </li><li> 10μMのCDG-CC（最終濃度）を追加します。 NOTE：（ 表2参照）（「ビーズ制御」という）CDG-CCない対照、および1mM c-ジGMP（「競合制御」）を補充した制御を含む。 注：CDG-CCの濃度は1〜10μMに調整することができる。 </li><li>暗闇の中で、回転ホイール上に少なくとも2時間（O / Nのための膜画分）、4℃でインキュベートする。 </li><li>短いスピンした後、架橋CDG-CCの反応性部分の活性化により4分間のUV光で、CaproBoxを使用して（λ= 310 nmの放射照度≥10ミリワット/​​ cm 2で、光源からの距離、物質一覧を見る= 2センチ）。注：ストリップ架橋前の蓋を外す。 </li><li>優しく均質化、25μlの5倍の洗浄緩衝液（5MのNaCl、250 mMトリス、pH7.5）を、よく再懸濁ストレプトアビジン磁気ビーズ50μlのを追加。 </li><li>回転ホイール上で1時間4℃でインキュベートする。 </li></ol> 6.洗浄ステップ注：（マグネット：材料リストを参照してください）​​。磁石を用いたPCRストリップ蓋の磁気ビーズの捕獲、始めます。その後、次の洗浄溶液を含む新しい1によりPCRストリップを交換してください。磁石を外し、ビーズを再懸濁し、2分間インキュベートする。スピンダウンし、新鮮な蓋によって蓋を交換してください。 <ol><li>洗浄工程（のみ可溶性画分） <ol><li> 200μlの1×洗浄バッファーで6回洗浄する。 </li><li> 200μlのHPLCグレードH 2 Oで一度洗浄する</li><li>洗浄液200μlの80％アセトニトリル中の6回。 </li><li> 200μlのHPLCグレードH 2 Oで2回洗浄</li></ol></li><li>洗浄ステップ（membraNE画分のみ） <ol><li> + 0.1％DDM200μlの1×洗浄緩衝液で5回洗浄する。 </li><li> 200μlの1×洗浄緩衝液+ 0.05％DDMに2回洗浄する。 </li><li> + 0.025％DDM200μlの1×洗浄緩衝液で一回洗浄する。 </li><li> 200μlの1×洗浄緩衝液+ 0.0125パーセントのDDMに一度洗ってください。 </li><li> 200μlの100mMのABCで3回洗浄し（炭酸水素アンモニウム、NH 4 CO 3）+ 2 M尿素。 </li></ol></li></ol> 7. MSサンプル調製<ol><li> 20μlの100mMのABC（膜分画用の100 mMのABC + 8 M尿素）で（直接蓋）ビーズを再懸濁し、1.5mlチューブに移す。 </li><li>膜画分は、500 rpmで振盪し、5分間60℃でインキュベートする。 </li><li> 0.5μlの200 mMのTCEP（トリス（2-カルボキシエチル）ホスフィン）を添加し、500 rpmで振盪、1時間60℃でインキュベートする。 25℃に冷却する。 </li><li>揺れ、ヨードアセトアミド新たに調製された400mmの0.5μl添加し、30分間25℃でインキュベート500回転数トン、暗所で。 </li><li> 0.5μlの0.5 M のN -アセチルシステインを追加し、500 rpmで振盪、10分間25℃でインキュベートする。 </li><li>膜画のみ：1μLのLys-Cを追加し、37℃、O / Nでインキュベートする。 </li><li> 2μgのトリプシンを追加し、500回転（乾燥を防ぐためにパラフィルムでラップ）で振とうし、37℃でO / Nインキュベートする。 注：サンプルは、この段階で-20℃で保存することができる。 注：メンブレン分のみ：トリプシンの添加の前に&lt;2 Mに尿素濃度を調整するために100 mMのABCを追加します。 </li><li>簡単に言えば、チューブをスピンダウンし、磁石でビーズを収集します。 </li><li>新しい1.5 mlチューブに上清を移し（ビーズが残っている場合は、この手順を繰り返します）。 </li><li> 5μlの5％TFA（トリフルオロ酢酸）+ 1μlの2 M塩酸（膜分画のための15μlの5％TFA + 5μlの2 M HCl）を追加します。 </li><li>条件C18をMicroSpinカラム150μlのアセトニトリル（ネストグループ、MA、USA）（2400 rpmで20秒スピン）。 </li><li>エクイ150μlの0.1％TFA（スピン20秒、2400 RPM）でC18カラムを2回librate。 </li><li>サンプルをロードし、2分、2000rpmでスピン。 </li><li>カラムにフロースルーをリロードし、紡糸工程（2分、2000 rpm）を繰り返します。 </li><li> 150μlの0.1％TFA、5％アセトニトリル（20秒、2400回転）で3回洗浄する。 </li><li>新しいチューブに取り、150​​μlの0.1％TFA、50％アセトニトリル（2分間、2000 rpmで）で2回溶出する。 </li><li>スピードバック中のペプチドを乾燥させる。 </li><li> 40μlの98％H 2 O、2％アセトニトリル、0.15％ギ酸に再懸濁。 </li><li>超音波処理20秒（パルス周期0.5、振幅100パーセント、物質一覧を参照）、5秒、回転数12,000rpm（ベンチトップ遠心機）をスピンダウン。ボルテックス10秒、及び5秒、12,000rpmでスピンダウン。 LC-MS / MS分析のためにHPLCバイアルに移す。 </li><li> -20℃で凍結する。 注：サンプルは、この段階で-20℃で保存することができる。 </li></ol> 8. LC-MS / MS分析<ol><li>ナノLCを実行します（ナノLCシステム）equippRP-HPLCカラム（75ミクロンインチx 37 cm）のC18樹脂を充填したと編（マジックC18 AQ3μm）を、95％溶媒A（0.15％ギ酸、2％のアセトニトリル）、5％の溶媒Bの直線勾配を使用して（ 0.2μlの/分の流速で60分間かけて35％溶媒B 98％アセトニトリル、0.15％ギ酸）。 </li><li> LC-MS / MS（エレクトロスプレーイオン源に接続されたデュアル圧力LTQ-オービトラップVelos質量分析計）を用いてペプチドを分析する。 注：データ収集モードは、MSが20の最も強いイオンを線形イオントラップにMS / MSスキャン、続い6万FWHMの分解能で質量分析計のFT部分をスキャンつの高解像度が得られるように設定した。 MS / MSの試みの効率を高めるために、荷電状態スクリーニング様式が割り当てられていない除外することが可能と単独イオンを充電した。前駆体は100イオン数を超えると共謀誘起解離がトリガされました。動的排除時間を30秒とした。イオン蓄積時間は300ミリ秒（MS）と50に設定したミリ秒（MS / MS）。 </li></ol> 9.データベース検索<ol><li> P.をダウンロード緑膿菌 NCBI-データベースNCBIのホームページを経由して（ <a href="http://www.ncbi.nlm.nih.gov/">http://www.ncbi.nlm.nih.gov/</a> ）。 </li><li> MassMatrix変換ツール（使用してマスコットジェネリックファイルにMS生のスペクトル（MGF）に変換<a href="http://www.massmatrix.net/mm-cgi/downloads.py">http://www.massmatrix.net/mm-cgi/downloads.pyを</a> ）。 </li><li> P.に対してMASCOTバージョン2.3を使用してこのMGFファイルを検索フォワード含む緑膿菌 NCBIデータベースと逆デコイタンパク質のエントリ。 </li><li> （プロリンが続く限り）2が完全にトリプシンペプチドで切断を逃し耐えリジンとアルギニンの後シリコトリプシン消化で実行します。 </li><li>固定修飾などのシステイン残基の変数の修正及びカルボキシアミドメチル（57.021464ダ）として酸化されたメチオニン（15.99491 DA）を可能にするために、データベースの検索パラメータを設定します。 MASCOTは私たちを検索するための高解像度スキャンをる、15 ppmまで前駆質量許容値を設定し、0.6 DAにフラグメント質量許容値を設定。最後に、1％タンパク質FDRを設定します。 </li><li> P.のマスコット検索をインポート足場（Proteomesoftware、バージョン3）に緑膿菌 CCMS実験は、1％のタンパク質FDR近くを取得し、合計スペクトルカウントを抽出するためのパラメータを設定する。 </li><li>本稿の代表的な結果のために、我々は競合制御と実験を比較するために対応のあるt検定を使用し、0.1以下のp値のみと考え安打、及び2以上のスペクトルカウント比（スペクトルカウント/競合制御スペクトルを試すカウント）。 </li><li>データは、さらなる分析のために任意の表計算ソフトにエクスポートすることができる。 </li></ol> 10.ラベルフリー定量<ol><li> Progenesis LC-MSソフトウェア（非線形力学、バージョン4.0）にRAWファイルをインポートします。 </li><li>デフォルトセッティにおけるLC-MSアライメントおよび特徴検出を行うNGS。 </li><li> Progenesis LC-MSからMGF形式でデータをエクスポートします。 </li><li> NCBI P.に対してMASCOTを使用して、MS / MSスペクトルを検索するフォワード含む緑膿菌データベースと逆デコイタンパク質のエントリ。 </li><li> Progenesis LC-MSへのデータベースの検索結果をインポートし、MS1機能へのペプチドの識別をマップします。 </li><li> （0.1以下のq値 、2上記のスペクトルカウント比閾値）データ評価（有意レベルの計算、倍変化を比）がSafeQuantのProteinSQAnalysisスクリプトが使用された。 </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radial+capillary+action+of+ligand+assay+for+high-throughput+detection+of+protein-metabolite+interactions.">Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 15528-15533 (2011).</li><li>DeSantis, K., Reed, A., Rahhal, R., Reinking, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+differential+scanning+fluorimetry+as+a+high-throughput+assay+to+identify+nuclear+receptor+ligands.">Use of differential scanning fluorimetry as a high-throughput assay to identify nuclear receptor ligands.</a> Nuclear receptor signaling. 10, e002 (2012).</li><li>Seidel, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+thermophoresis+quantifies+biomolecular+interactions+under+previously+challenging+conditions.">Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.</a> Methods. 59, 301-315 (2013).</li><li>Merighi, M., Lee, V. T., Hyodo, M., Hayakawa, Y., Lory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+messenger+bis-(3&#8216;-5&#8217;)-cyclic-GMP+and+its+PilZ+domain-containing+receptor+Alg44+are+required+for+alginate+biosynthesis+in+Pseudomonas+aeruginosa.">The second messenger bis-(3&#8216;-5&#8217;)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa.</a> Molecular Microbiology. 65, 876-895 (2007).</li><li>Qi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+cyclic+diguanylate+in+the+non-catalytic+EAL+domain+of+FimX+induces+a+long-range+conformational+change.">Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change.</a> J. Biol. Chem. 286, 2910-2917 (2011).</li></ol>

The authors have nothing to disclose.

特別な注意は、プロトコルのいくつかのステップで撮影する必要があります。タンパク質濃度は10 mgの濃度の重要なパラメータである/ mlの細胞が特定の増殖条件（ 例えば、バイオフィルムまたは小型コロニー変種）で増殖させたときに到達することが困難である。従って、ペレットの再懸濁を溶解緩衝液の少量で行われるべきである。タンパク質濃度は8mg / mlまで減少させることがで?...

c-ジGMPは、その成長と行動の様々な側面を制御するために、ほとんどの細菌によって使用される重要なセカンドメッセンジャーである。例えば、c-ジGMPは、細胞周期進行、運動性およびエキソポリサッカライド及び表面アドヘシン2-4の発現を調節する。このようなプロセスのc-ジ-GMPの協調を通じて、バイオフィルム形成、病原菌5の範囲の慢性感染症に関連付けられているプロセスを促進する。 c-ジ-GMPは、触媒GGDEFドメイン4を保有するジグアニルシクラーゼ（DGCs）と呼ばれる酵素によって合成されている。いくつかのDGCsはダウンc-ジ-GMP結合の際シクラーゼ活性を調節阻害部位を有する。 c-ジ-GMPの劣化は、触媒EALまたはHD-GYPドメイン6,7のいずれかを保有するホスホジエステラーゼの二つの異なるクラス（偏微分方程式）によって触媒される。 直接c-ジGMPに結合する既知のエフェクタータンパク質の大部分は、タンパク質の唯一の3つのクラスに属する：触媒を同盟国、非アクティブGGDEFまたはEALドメインとピルツドメイン、c-ジ-GMPが8と結合すると立体構造変化を受ける小さな分子スイッチ。 DGCs、偏微分方程式とピルツタンパク質が十分に特徴付けされ、そのドメインは比較的安全にin silicoで予測することができる。特に関心は現在c-ジ-GMPエフェクターの新しいクラスの識別に焦点を当てている。別の結合モチーフを有するいくつかのc-ジ-GMPのエフェクターは、 バークホルデリアcenocepacia中のCRP / FNRタンパク質ファミリーBcam1349またはPにおける転写調節因子FleQとして最近、記載された緑膿菌 9,10。また、c-ジGMP特異的リボスイッチは、最近同定され、c-ジGMP依存性の様式11に遺伝子発現を制御することが示された。異なるエフェクターのc-ジ-GMPが結合モチーフはわずかしか保存されたようなタンパク質の生物情報学の予測を困難にしている。この問題に対処するために、我々は、c-ジGMPのSPEの使用に基づく生化学的方法を開発cific捕獲化合物は、質量分析法1,12,13と組み合わせる。 我々は最近、（CDG-CC、 図1）は、新規三価のc-ジ-GMPの捕獲化合物を設計しています1。 、c-ジ-GMP結合タンパク質を捕捉するために餌として使用1）c-ジGMP部分結合したタンパク質にCDG-CCをリンク交差するために使用する2）紫外線光活性化反応基と：この化学足場はで構​​成されている3）ビオチンは、ストレプトアビジン被覆磁気ビーズを用いて捕捉されたタンパク質を単離する。 CDG-CCは、直接かつ特異的に細胞溶解物のような高分子の複雑な混合物からc-ジGMPエフェクターを捕捉するために使用することができる。捕獲化合物系、化学プロテオミクスに基づくアプローチは、以前は、広範囲の生物に適用可能であることが報告されており、 例えばカウロバクターcrescentus、サルモネラ·エンテリカ血清型ネズミチフス菌およびP.緑膿菌 1,14。 この方法論の論文では、我々は、P。の抽出物を使用してCCMS手順の深さの説明で提供例として緑膿菌 。この研究は、生化学的に、小分子のシグナル伝達に関与する新規成分を同定するための強力で汎用性の高いツールとしてCCMSを確立します。

<table border="0" cellpadding="0" cellspacing="0" width="1179">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:236px;">caproBox</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">1-5010-001 (220 V)</td>
			<td style="width:515px;">UV lamps coupled to a cooling 96-plate cooling block, for the photoactivation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">caproMag</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">included in the CCMS Starter Kit</td>
			<td style="width:515px;">For easy handling of magnetic particles without pipetting</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:236px;">c-di-GMP caproKit</td>
			<td style="width:223px;">caprotec bioanalytics</td>
			<td style="width:207px;">upon request</td>
			<td style="width:515px;">The kit contains the c-di-GMP-capture compound, c-di-GMP (for the competition control), streptavidin coated magnetic beads, capture buffer, and washing buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:236px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:223px;">GE Healthcare</td>
			<td style="width:207px;">17-0851-01&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">12-tube PCR strips</td>
			<td style="width:223px;">Thermo Scientific</td>
			<td style="width:207px;">AB-1114</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UIS250v sonicator with VialTweeter</td>
			<td style="width:223px;">Hielscher ultrasound technology</td>
			<td style="width:207px;">UIS250v and VialTweeter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Miniature French Pressure Cell</td>
			<td style="width:223px;">Thermo Electron Corporation</td>
			<td style="width:207px;">FA-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

capture compound mass spectrometry - a powerful tool to identify novel c-di-gmp effector proteins

かなりの進歩は、第二メッセンジャーc-ジ-GMPの合成（ジグアニルシクラーゼ）及び分解（ホスホジエステラーゼ）に関与する酵素の同定および特徴に向けた最後の十年の間に行われている。これとは対照的に、ほとんど情報がこのシグナル伝達分子は、細胞プロセスの多様な範囲を規制を通じて分子機構と細胞成分に関する利用可能です。知らエフェクタータンパク質のほとんどは、ピルツファミリーに属するか、触媒作用をあきらめているとエフェクター機能を採用しているジグアニルシクラーゼ又はホスホジエステラーゼを縮退している。したがって、より良い細菌実験方法の広範囲の細胞c-ジGMPネットワークを定義することは失敗するインシリコ予測における信頼性のあるもののための新規のエフェクターを識別し、検証する必要がある。 我々は最近の強力なツールとしての新規捕獲化合物質量分析法（CCMS）に基づく技術を開発しました生化学的にc-ジ-GMP結合タンパク質を同定し、特徴づける。この技術は、以前に生物1の広い範囲に適用可能であることが報告されている。ここでは、我々はそのようなシグナル伝達成分を調べるために利用するプロトコルの詳細な説明を与える。例として、 緑膿菌 、c-ジ-GMPが病原性とバイオフィルム制御に重要な役割を果たしている日和見病原体を使用しています。 CCMSは、c-ジ-GMPネットワークの既知または予測コンポーネントの74パーセント（51分の38）を同定した。この研究は、詳細にCCMS手順を説明し、小分子のシグナル伝達に関与する新規成分を同定するための強力で汎用性の高いツールとしてそれを確立します。

P.における新規c-ジ-GMPのエフェクターを同定するために、 緑膿菌我々は体系的にPの可溶性および膜画分を分析するためにCCMSを使用対数期培養（OD 600 = 0.5）から緑膿菌菌株 PAO1。ここでは、この釣りの遠征の代表的な結果を要約し、議論する。四つの独立した生物学的なレプリカが使用された。各実験のために二つの異なるCDG-CC濃度（5μMおよび10μM）を用い...

Watch this Scientific Journal Video about Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins at JoVE.com

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

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 ...

capture-compound-mass-spectrometry-powerful-tool-to-identify-novel-c

Research

JoVE Journal

Biology

11.5K ビュー.  Biozentrum of the University of Basel. 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.

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

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

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

P2X2リガンド依存性陽イオンチャネルと相互作用するタンパク質を特定するためのプロテオミクス

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

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

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

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

エレクトロスプレーイオン化質量分析法を用いて酵母Lipidomeの定量評価

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

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

スプリットユビキチンによる膜酵母ツーハイブリッド（神話）システム：タンパク質間相互作用を識別するための強力なツール

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

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 ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR -クリップ - RNA結合タンパク質のトランスクリプトーム全体の結合部位を同定する方法

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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 ...

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 ...

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

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

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 ...

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 ...