SWとTMVは、国立衛生研究所のNCRRとNHLBIでサポートされています。 BSKとHSは、国立衛生研究所のNINDSとNIGMSでサポートされています。

実験手順実験手順（図2）は以下の段階的な方法で説明されている4つの部分から構成されています。 パート1：P2X2受容体のC末端のサブクローニングおよび発現。 我々はそれが結合する脳のタンパク質を同定するために全長細菌におけるP2X2受容体のC末端を表明している。 <ol><li> P2X2受容体のC末端（残基353〜472）（図1）pGEX 4NT1（GEライフサイエンス）にクローン化と塩基配列決定と検証、PCRにより増幅した。 </li><li>組換えプラスミドを大腸菌に形質転換された組換えタンパク質の発現用大腸菌（BL21）。 </li><li> P2X2 CT - GSTプラスミドを含む細菌（ 大腸菌 BL21）のグリセロールストックを滅菌ピペットの先端で擦り取り、適当な選択マーカーとルリア-ベルターニ（LB）培地（50 g / mlのアンピシリン）5mlに接種し、 37℃でオービタルシェーカーで一晩インキュベート。 </li><li> 600nmの光学密度に達するまで一晩培養したが、適切な抗生物質（50μg/ mlのアンピシリン）を含むLB培地250 mlに接種し、37℃で2〜3時間、250 rpmでオービタルシェーカーでインキュベートした〜 0.6から0.7。 </li><li>培養は、1mMのIPTGで誘導し、さらにタンパク質発現のための3時間37℃でインキュベートした。 </li><li>文化は、その後、4C（文化1mlのは、発現レベルを分析用に保存し、&quot;誘導&quot;としてラベル付けされた）で15分間5000gで回転させた。上清を捨て、ペレットを生理食塩水トリス- EDTA（STE）緩衝液（10mMトリス- HCl、150mMのNaCl、1mMのEDTA pH8.0）を20mlに再懸濁した。 </li><li>懸濁液を50mlファルコンチューブに15分間5000gで回転させた。上清を捨て、半乾燥したペレットは、一晩、-70℃で凍結した。 </li><li> - 70Cペレットを氷冷溶解緩衝液40ml中（2％（w / v）のサルコシル、15 mgのリゾチーム、150mMのNaCl、50mMのトリスpH7.5、5mMのDTTと完全なプロテアーゼ阻害剤の錠剤）に再懸濁し、 、および懸濁液を氷上で30分間インキュベートした。このインキュベーションの間、3ミリリットルグルタチオン - セファロース4BビーズをPBSに続いてリン酸緩衝生理食塩水（PBS）、20mlのT - PBS（0.1％（v / v）のPBSでtritonX - 100）20mlで洗浄した。ビーズを1mlのPBSに再懸濁させた。 </li><li>細菌懸濁液は、液体窒素と超音波処理で3回凍結融解（オフ/オン1秒、氷上で3分間30ミクロン）と、さらに4％（v / v）のトリトンX - 100、10mMのマグネシウムと30分間インキュベートした。氷上で4と2 mMのATP。 </li><li>ライセートは、4Cとペレットで15分を20000 gで遠心し（上清のペレットと1 mlのサンプルが細胞質ゾルと膜中のタンパク質の量をテストするためにSDSのページ用に保存された）を廃棄した。上清を4℃で1時間（または一晩）のためのビーズとインキュベートした。 </li><li>ビーズを5分間、500gで遠心した上清を（サンプルの非結合画分のために保存された）を廃棄した。ビーズをT - PBSに5回（洗浄は洗浄のコントロールのために保存された）で洗浄した。 </li><li>ビーズを50％（w / v）のスラリーを得たPBSに再懸濁し、使用まで4℃で冷蔵庫に保存されていました。タンパク質濃度はBradfordアッセイ（製造者の指示当たり）によって推定された。 </li></ol>第2部：全脳ライセートの調製 P2X2受容体が豊富に脳8で表されることが知られている。本解析では、我々は、ラットの全脳溶解（図3）から、P2X2受容体と相互作用するタンパク質を特定しようとした。 <ol><li>成体ラットの脳を採取（動物はUCLA IAACUC承認されたプロトコルに従って、実験動物の管理と使用に関するNI​​Hのガイドに従って屠殺し）し、直ちに氷冷PBS（3X）を洗浄した。 </li><li> 150mMのNaClを含有する氷冷溶解バッファーのセル10ミリリットル（収穫脳の〜5倍の湿重量）を溶解するために、50mMトリス- HCl、pH7.4の、10mMのEDTA、1mMのEGTA、1mMのNaFを、1mMののNa 3 VO 4、1mMのPMSF、ロイペプチン、5μg/ mlの、1％（v / v）のNP - 40、およびプロテアーゼインヒビターカクテル錠を収穫し、脳に追加された。 </li><li>均質な一貫性の混合が達成されるまで脳組織をガラスDounceホモジナイザーで氷の上均質化した。 </li><li>細胞溶解液は、連続したセルを削除するには、5分間に2000 gで回転させた。上清はそのまま細胞小器官と細胞膜の凝集物を除去するために60分間29000グラムで再び採取し、分離独立しました。 </li><li>すぐに遠心分離後上清を新しいチューブに移した。全脳のライセートのタンパク質濃度をブラッドフォードアッセイにより測定した（通常〜15 mg / mlの）。 </li><li>ライセートを分取し、AT - 70Cで保存した。 </li></ol>パート3：P2X2 C -テイル関連したタンパク質の単離全脳ライセートからP2X2 CT - GSTの結合パートナーを同定するために、アッセイのプルダウンは、CT - Gを用いて実施したSTは、餌としてグルタチオンセファロース4Bビーズに固定化。コントロールについては、ビーズに結合しただけではGSTが使用されました。 <ol><li>脳ライセートを4℃で1時間、GSTビーズとpreclearedした。ペレットを廃棄した上清を更なる分析のために使用されていました。 </li><li>グルタチオンセファロース4 Bのビーズに結合したGSTとP2X2 CT - GST（10μg）を等量のLysis Buffer中で4℃で回転して、ラットの全脳のライセートからprecleared可溶化タンパク質を100gで一晩インキュベートした。 </li><li>ビーズは5分間1000gで遠心した上清を捨てた。 </li><li>ビーズを溶解緩衝液で一回洗浄し、修正Laemliバッファーで煮沸された[4％（w / v）のSDS、10％（v / v）の2 - メルカプトエタノール、2％（v / v）グリセロール、0.004％（W / V）ブロモフェノールブルーおよび0.125 M TrisCl pHを6.8] 5分間。 </li><li>サンプルは、5分間5000gで遠心し上清は、（図4）10％SDS - PAGEにより分離した。ゲルを染色SYPROルビータンパク質ゲルを染色（製造元の指示に従って）と紫外線下で可視化した。 P2X2 CT -関連タンパク質を餌としてGST - nullを使用して回復したものと比較した。四生物学的には（四脳およびつの独立したプルダウンをすなわち）が実行された複製。 </li></ol>パート4：タンパク質の同定。 ゲル電気泳動により分離されたタンパク質をゲルから切り出し、質量分析12で同定された（注）。過程を通して粉塵がない場合は、ケラチン汚染を減らすために重要です。実験者は、常時、ヘアネット、手袋を顔のマスクを着用し、手袋を使用することなく関心のゲル地区には決して手を触れないようにしてください。 <ol><li>ダウンP2X2プルダウンから回収されたすべての可視タンパク質バンドは、きれいなカミソリの刃とさいの目に切られた（図4）を摘出した。 GST -ヌルのゲルの対応する領域は、車線プルダウンものレベルより下のタンパク質のその存在を保証するためにバンドの染色（GST - nullの餌のための特異的なバンドがまた摘出したと確認された）に関係なく同じ方法で摘出染色感度は見逃していなかった。 </li><li>ゲル片を200 50％50 mM重炭酸アンモニウムの溶液（v / v）のアセトニトリル（ACN）と室温で15分間ボルテックスチューブをインキュベートした。この洗浄ステップを繰り返します。 </li><li>ゲル片は、200μlのアセトニトリル（ゲル片が縮小し、色で不透明になるはず）を追加することにより脱水した。 </li><li> Acetontrileは削​​除され、ゲル片は、約10分間speedvacで乾燥させた。 </li><li>ゲル片はその後、新たに調製した10mMのDTT/10 mMトリス（2 - カルボキシ - エチル）ピースを浸漬するのに十分な量のホスフィン塩酸塩（TCEP）の30μlのでインキュベートした。これは、56C水浴で30分間放置した。 </li><li>次のDTT溶液を50％アセトニトリル溶液とammounium炭酸で洗浄ゲルスライスに100μlの500 mM重炭酸アンモニウムに置き換えられました。 </li><li>洗浄溶液を吸引し、アセトニトリル200μlのは、ゲルを脱水に追加されました。 </li><li>アセトニトリルを除去し、100μlを調製したばかりの100mMヨードアセトアミドのソリューションは、フリースルフヒドリルをアルキル化するために追加されました。このステップでは、暗所で室温で25分間進めた。 </li><li>ヨードアセトアミド溶液を除去し、ボルテックスしながらゲル片は、2分間50％acetontrile液（pH8.0）に50 mM重炭酸アンモニウム200μlで洗浄した。この洗浄工程を繰り返した。 </li><li>洗浄溶液を除去した後、ゲル片を〜10分間speedvacによって削除された200μlのアセトニトリルと共にインキュベートした。 </li><li>このステップでゲル片は、トリプシン消化のための準備が整いました。ゲル片を30μlのトリプシン溶液（20 ng /μLのワーキング濃度）中でインキュベートしても腫れするゲルのための10分間氷上に置いた。過剰トリプシンは37℃で12〜16時間進行させた彼らが消化を通して浸漬保つために50mMの重炭酸アンモニウムと30μlのを重ねてゲル粒子を除去し、再水和した。 </li><li>五の5％溶液（v / v）のギ酸はトリプシンと逮捕消化を無効にするには追加されます。 </li><li>チューブは〜15分間攪拌し、きれいと標識0.5mlのバイアルに移していたチューブの底に液体をもたらすために簡単に遠心分離した。 </li><li>それらはちょうど簡単な遠心分離によって分離、10〜15分間2回ボルテックスで続いてカバーされたので、50％アセトニトリル中0.1％の次の30μL（v / v）のギ酸はゲル片に追加されました。このステップは、ステップ間のギ酸 - アセトニトリルを置き換えて、一回繰り返した。 </li><li>最終的な液体は、単一バイアルで除去し、すべての抽出溶液は、混合した。このボリュームはspeedvacで〜30 ULに減少した。サンプルは現在、逆相質量分析法によるナノ液体クロマトグラフィーと蛋白質の同定のための準備ができていた。この特定の例では、このステップはThermoFからOrbiトラップタンデム質量分析計で実施され他のモデルとメーカーの楽器は簡単にこれまでに説明したアプローチと相まってすることができるがisher（その高質量精度、タンパク質の検出と一般的に堅牢なオペレーティング機能のための高速なデューティサイクルのために選択）、。 </li><li>質量分析の詳細と基準について簡単に紹介されています。すべてのペプチドは逆相ナノLC（Eksigent）によって分離し、ペプチドを3μL/ minの流量でC18逆相カラム上に負荷された。移動相0.1％ギ酸と水で2％ACNでした、移動相Bは0.1％ギ酸とACNで20％の水である。ペプチドは、220 NL / minは5分以上95％Bにして、90分間にわたって50％Bに5％のBからの線形グラデーションを使用しての流速でカラムから溶出させ、そして最終的に5分間一定の95％Bを保っていた。スペクトルは、MS / MSスキャンのMSのスキャンとLTQに使用Orbiトラップとデータ依存モードで獲得されました。ペプチドはBioworksのソフトウェアパッケージに統合SEQUESTのアルゴリズムを用いてラットIPIのデータベースv.3.53に対してスペクトルを検索することによって同定された。 xcorrは≥2（+1）、≥3（+2）、≥4（+3）、およびDeltaCN&gt; 0.1：それぞれのペプチドは、以下の基準を満たしていた。タンパク質同定のために使用される全てのスペクトルは、手動で検査し、以下の二つのペプチドが同定されたときにタンパク質が受け入れられなかった。唯一のタンパク質はGST -ヌルプルダウン中に4 P2X2プルダウンのそれぞれと欠席以下の提示は、さらなる分析のために考慮された。 </li></ol><img src="/files/ftp_upload/1178/1178fig1.jpg" alt="図1" /> 図1。 P2X2受容体のサブユニットの模式図。 A.漫画は、P2X2受容体のサブユニットのトポロジを示します。細胞質ドメインはNおよびC末端で構成されています。 P2X2受容体（赤）のC末端は、アッセイをプルダウンするための餌として使用した。本研究で使用したP2X2受容体C末端のB.アミノ酸配列は。 <img src="/files/ftp_upload/1178/1178fig2.jpg" alt="" /> 図2。発現、精製に使用されるプロトコルのフローチャートとタイムライン、プルダウンし、タンパク質の同定。我々は、タイムラインとプロトコルの概要を示す。成体ラット脳ライセートはアッセイプルダウンに使用する直前に新しく調製した。 <img src="/files/ftp_upload/1178/1178fig3.jpg" alt="" /> 図3。ラット脳におけるP2X2 C末端ネットワークのバイナリプロテオーム解析の模式図。GSTビーズに結合したGST蛋白質と融合させたP2X2受容体のC末端は、アッセイをプルダウンするために使用されました。脳ライセートを調製し、タンパク質は固定化した組換えタンパク質とインキュベートした。非結合画分を溶解緩衝液で洗浄した。タンパク質は、質量分析により同定した。 <img src="/files/ftp_upload/1178/Fig%204%20Singh%20et%20al.jpg" alt="" /> 図4。P2X2受容体のC末端の結合パートナーの識別。GST（青い箱）と融合受容体のC末端と相互作用推定タンパク質のスペクトルを示すSYPRO染色したゲル。単独でGST単独（黄色のボックス）とグルタチオンセファロースビーズ用コントロールレーンも表示されます。矢印は質量分析によって、さらなる分析のために摘出しユニークなバンドの例を示している。

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イオンチャネルは、膜内在性タンパク質の主要なクラスです。彼らは選択的に形質膜を横断し、その電気化学的勾配下にイオンの移動を可能にする水の満たされた気孔が含まれています。開いた状態および閉じた状態との間のイオンチャネルのゲート。ゲーティングのステップは、P2Xリガンド依存性イオンチャネルの場合の送信機（例：ATP）によって引き起こされる、またはそれが他のタン?...

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<td>Acetonitrile</td>
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<td>Acrylamide</td>
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<td>Ampicillin</td>
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<td>Ammonium Bicarbonate</td>
<td>Reagent</td>
<td>Fluka</td>
<td>09830</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Persulphate (APS)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3678</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine Triphosphate (ATP)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7699</td>
<td>&nbsp;</td>
<tr>
<td>Bradford reagent</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>B-392</td>
<td>&nbsp;</td>
<tr>
<td>Commassie blue R-250</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<td>Sc-24972</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotritol (DTT)</td>
<td>Reagent</td>
<td>EMD</td>
<td>3860</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>Reagent</td>
<td>VWR</td>
<td>VW1474-01</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene Glycol tetraacetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E8145</td>
<td>&nbsp;</td>
<tr>
<td>Formic acid</td>
<td>Reagent</td>
<td>EMD</td>
<td>11670-1</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione Sepharose 4B beads</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-5132-01</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric acid (HCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1758</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>15502</td>
<td>&nbsp;</td>
<tr>
<td>Iodoacetamide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I1149</td>
<td>&nbsp;</td>
<tr>
<td>Luria-Bertani (LB) Media</td>
<td>Reagent</td>
<td>EMD</td>
<td>1.00547.5007</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L8511</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>Reagent</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Sulphate (MgSO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7653</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Flouride (NaF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S7920</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Orthovanadate (Na3VO4)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S6508</td>
<td>&nbsp;</td>
<tr>
<td>Nonidet P40</td>
<td>Reagent</td>
<td>Fluka</td>
<td>74385</td>
<td>&nbsp;</td>
<tr>
<td>Phenylmethanesulphonylfluoride (PMSF)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Protease inhibitor tablet</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8820</td>
<td>&nbsp;</td>
<tr>
<td>Protein standard </td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>161-0305</td>
<td>&nbsp;</td>
<tr>
<td>Sarkosyl</td>
<td>Reagent</td>
<td>Acros</td>
<td>61207</td>
<td>&nbsp;</td>
<tr>
<td>Screw top vial</td>
<td>Tool</td>
<td>Agilent Technologies</td>
<td>5182-0866</td>
<td>&nbsp;</td>
<tr>
<td>Sodium dodecyl sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>L4509</td>
<td>&nbsp;</td>
<tr>
<td>SYPRO&#174; Ruby protein gel stain</td>
<td>Reagent</td>
<td>BIO-RAD</td>
<td>170-3125</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N',N'-Tetramethylethylenediamine (TEMED)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9281</td>
<td>&nbsp;</td>
<tr>
<td>Tris base</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Promega</td>
<td>V5111</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5927</td>
<td>&nbsp;</td>
<tr>
<td>Water</td>
<td>Reagent</td>
<td>Burdick&Jackson</td>
<td>365-4</td>
<td>&nbsp;</td>
<tr>
<td>LTQ-Orbitrap tandem mass spectrometer</td>
<td>Tool</td>
<td>ThermoFisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nano Liquid Chromatography System</td>
<td>Tool</td>
<td>Eksigent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B-Mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6250</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

proteomics to identify proteins interacting with p2x2 ligand-gated cation channels

リガンド依存性イオンチャネルは神経系1でシナプスのコミュニケーションを貫く。システインループ、グルタミン酸ゲートおよびP2X受容体チャネル2：哺乳類ではリガンド依存性チャネルの3つのファミリがあります。それぞれの場合に送信機の結合はイオンが電気化学的勾配を流れに経由する細孔の開口部につながる。多くのリガンド依存性チャネルは、またダウンストリームチャネルの開口部の持続時間を超える可能性の役割5（例えば、遺伝子の調節）をシグナリングしているカルシウムイオン3、4、に透過性である。したがって、リガンド依存性チャネルは、数ミリ秒から数日までの幅広い時間スケールで信号を送ることができる。これらの重要な役割を考えるとそれはそれ自体がタンパク質、そしてどのようにこれらのタンパク質は、シグナル伝達に調整できることで規制されている方法リガンド依存性イオンチャネルを理解する必要があります。最近の研究では、多くが、すべてではありませんが、チャンネルは蛋白質シグナル伝達複合体6の一部である可能性を示唆している。この記事では、P2X2受容体細胞質ドメインのC末端の側面に結合するタンパク質を同定する方法を説明します。 P2X受容体はATP依存性陽イオンチャネルであり、7つのサブユニット（P2X1 - P2X7）で構成されています。 P2X受容体は広く彼らは興奮性シナプス伝達や神経伝達物質放出7のシナプス前促通を媒介する脳、で表されます。 P2X受容体は興奮性および非興奮性細胞に存在し、神経細胞シグナリング、炎症と心血管機能8で重要な役割を仲介している。 P2X2受容体は神経系9に豊富であり、この研究の焦点です。各P2Xサブユニットは細胞外領域7と細胞内のNおよびC末端（図1a）7によって分離された二つの膜貫通セグメント（TM1＆TM2）を持つと考えられている。 P2Xサブユニット10は、（P2X1 - P2X7）アミノ酸レベル11で30〜50％の配列相同性を示す。 P2X受容体はイオンチャネル型受容体の中で最も単純な化学量論であるのみ3つのサブユニットが含まれています。 P2X2のC -末端は120アミノ酸（図1b）を構成され、P2X2受容体のシグナル伝達複合体の一部である可能性があるという仮説を支持する、いくつかのタンパク質のドッキングのコンセンサス部位が含まれています。いくつかの機能がP2X2受容体9のない研究のC -末端に起因しているが、しかし、全長C末端を介して、このタンパク質の細胞内側に結合する分子のパートナーを説明しました。この方法を本稿では、P2X2受容体の全長C末端と相互作用するタンパク質を同定するプロテオミクスアプローチを説明します。

Watch this Scientific Journal Video about Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels at JoVE.com

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

我々は、ATP依存性P2X2受容体の全長のC末端に結合する脳の蛋白質を識別するための単純なプロトコルを記述する。すべてのP2X受容体へのこのアプローチの拡張と体系的なアプリケーションは、P2X受容体シグナル伝達の理解につながることが期待される。

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 ...

proteomics-to-identify-proteins-interacting-with-p2x2-ligand-gated

Research

JoVE Journal

Biology

14.6K ビュー.  David Geffen School of Medicine, University of California, Los Angeles. 我々は、ATP依存性P2X2受容体の全長のC末端に結合する脳の蛋白質を識別するための単純なプロトコルを記述する。すべてのP2X受容体へのこのアプローチの拡張と体系的なアプリケーションは、P2X受容体シグナル伝達の理解につながることが期待される。

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

生物学

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 ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Coregulated遺伝子の潜在的な規制モチーフを特定するためにスコープの使用

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

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 ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

成長因子Progranulinと相互作用するタンパク質を識別するように変更酵母ツーハイブリッドシステム

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

相互作用するリガンドおよび/またはアンタゴニストと、哺乳動物核内受容体残基を同定するために逆酵母ツーハイブリッドシステム

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Bottom-up and Shotgun Proteomics to Identify a Comprehensive Cochlear Proteome

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that transduce the mechanical energy of sound into an electro-chemical energy processed by the peripheral and central nervous systems. Several proteomic techniques have been developed to study the cochlear inner ear, such as two-dimensional difference gel electrophoresis (2D-DIGE), antibody microarray, and mass spectrometry (MS). MS is the most comprehensive and versatile tool in proteomics and in conjunction with separation methods can provide an in-depth proteome of biological samples. Separation methods combined with MS has the ability to enrich protein samples, detect low molecular weight and hydrophobic proteins, and identify low abundant proteins by reducing the proteome dynamic range. Different digestion strategies can be applied to whole lysate or to fractionated protein lysate to enhance peptide and protein sequence coverage. Utilization of different separation techniques, including strong cation exchange (SCX), reversed-phase (RP), and gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) can be applied to reduce sample complexity prior to MS analysis for protein identification.

Proteome analysis of the cochlear sensory epithelium can be challenging due to its small size and because membrane proteins are difficult to isolate and identify. Both membrane and soluble proteins can be identified by combining multiple preparative methods and separation techniques along with high-resolution mass spectrometry.

総合蝸牛プロテオームを識別するために、ボトムアップとショットガンプロテオミクス

Proteomics is a commonly used approach that can provide insights into complex biological systems. The cochlear sensory epithelium contains receptors that ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

細胞表面プロテオミクスのための糖ペプチドキャプチャ

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

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.