この作品は、イノベーションのためのカナダの財団、ゲノムカナダ、オンタリオゲノム研究所、イノベーションのオンタリオ州省、JGとAE赤発現大腸菌にヘルスリサーチ助成のカナダの協会からの資金によってサポートされていました大腸菌株DY330は、ドナルド·L·コート（米国国立がん研究所、フレデリック、メリーランド州）からの親切な贈り物だった。

1。 大腸菌における遺伝子固有のSPA-タギングの構築大腸菌 DY330ひずみ<ol><li> SPA-タグのDNA配列及びカナマイシン抗生物質耐性マーカーカセット（菅R）が包含プラスミドpJL148は、ポリメラーゼ連鎖反応（PCR）増幅7のテンプレートとして使用されています。 27 BP（5&#39;-AGCTGGAGGATCCATGGAAAAGAGAAG -3 &#39;）タグ特異的なフォワードプライマーとフレーム内の標的遺伝子の終止コドンのすぐ上流に位置する45 ntの遺伝子特異的なフォワードプライマー、および45 ntの遺伝子特異的リバースプライマーは、すぐに位置して94：27 BP（5&#39;-GGCCCCATATGAATATCCTCCTTAGTT -3 &#39;）タグ特異的リバースプライマーを用いたフレーム内の標的遺伝子の終止コドンの下流には、以下のPCRサイクリング条件を用いpJL148からSPAタグと菅Rのカセットを増幅するために使用される5分間℃、1分間、68で1分間、55°C、10分間68℃に続いて2分10秒の場合はC、94°Cの30サイクル。これらの増幅された配列は、サーブ異所的に導入されたλ-赤相同組換え用機械の基板（下記参照）。 </li><li> PCR産物は、エレクトロポレーションを妨げる可能性が塩を除去するためにQIAQUICK PCR精製キットを用いて精製する。精製したPCR産物は、その後、λ-赤組換え機構が10を発現しているDY330株における特定の遺伝子の3 &#39;末端（ネイティブ終止コドンのすぐ上流側）に統合することをターゲットにしています。 </li><li> DY330λ - レッド発現株を180 rpmで振とうすることにより、32℃で2ミリリットルルリア - ベルターニ（LB）培地中で一晩増殖されています。一晩培養した培養液の1ミリリットルは続いて500ミリリットルの三角フラスコに70 mlの新鮮なLB培地に接種する。接種は、OD 600が約0.8に達するまで、180 rpmで振とうすることにより、32℃で栽培されています。 </li><li>文化は、穏やかに振とうすることにより細胞を42℃の水浴中でフラスコをインキュベートすることによって誘導される新鮮な250ミリリットルの三角フラスコ、°Cに転送されます15分間180回転。すぐに誘導した後、フラスコを振とうしながら、少なくとも30分間氷水スラリー浴でインキュベートする。 </li><li>氷のように冷たい文化は直ちに予冷50mlポリプロピレンチューブに移し、4℃で6分間3993 xgで遠心分離にかけ℃です。細胞ペレットを50mlの氷冷滅菌水に再懸濁し、4℃で6分間3993 xgで再び遠心分離する細胞ペレットを冷水1mlに再懸濁し、1.5ミリリットルEffendorfチューブに移し、4℃で20秒間、最大速度で遠心分離する氷冷水で2または3回の洗浄工程後、細胞ペレットを電気コンピテントセルで、その結果、氷冷滅菌水700μlにようやく再懸濁する。 </li><li>エレクトロポレーションにより、精製されたアンプリコンの1μlをエレクトロコンピテントセル40μlの中に導入される。 2.5kVで、パルスで25μF：細胞は、次の設定を使用してバイオ·ラッドGenePulser IIに​​エレクトロポアール200Ωのコントローラ。相同組換えと統合した後、正常染色体へのタグ/カセットを換え形質転換体は、菅（ 図1A）に対する耐性に基づいて選択されます。複数の形質転換体はウェスタンSPA-タグのFLAGエピトープに対する選択的な抗FLAG M2抗体によるSPA-タグ融合タンパク質の正しい世代（ すなわち、正信号）を確認するためにブロッティング用に選択されます。 </li><li>確認されたカルボキシ末端のSPA-タグ融合株は、SPA精製プロトコルを使用して採取された細胞から可溶性タンパク質複合体を単離するために、大規模で培養する。アフィニティータグ精製操作に必要な手順の概要を図1Bに示されている。 </li></ol> 2。培養すると超音波処理<ol><li> SPA-タグE.100μlを接種する50ミリリットルTerrificブロス（TB）に大腸菌グリセロールストックは、液体培地は、25μg/ mlのOの50μlを補充250ミリリットルの三角フラスコ中のf菅ソリューションを提供します。 32℃で一晩培養する 注：ひずみDY330の温度誘導性λ赤カセットは温度感受性リプレッサー10の制御下にあるので、SPA-タグ大腸菌株は、32℃で栽培されている </li><li> 4リットルのフラスコに菅の25μg/ mlのを補足した990ミリリットル新鮮結核に一晩培養液10ミリリットルを転送します。 OD 600が約2〜3に達するまで培養は、32℃で一定には5〜6時間、250rpmで振盪しながら成長させる。 </li><li> 1リットルEを転送大腸菌は、SPA-タグ·文化遠心ボトルをきれいにし、15分間ベックマン℃·J6-HCの遠心分離機@ 3993×gで4℃で細胞を回転させる。 </li><li>上清を遠心分離ボトルから削除されます。 大腸菌を懸濁する超音波処理緩衝液25mlを大腸菌細胞ペレット。すべての回で、氷上で遠心ボトルを維持することを確認します。 </li><li>再懸濁ペレットです50ミリリットルポリプロピレンファルコンチューブに移し、液体窒素を用いて凍結した。凍結細胞は-80℃で長期使用するために保存されます。 </li><li>超音波処理に先立って、氷の上にペレットを保つことによって完全に凍結細胞を解凍します。解凍されたサンプルは、超音波処理のために氷上に置い滅菌ステンレス製のカップに移しています。プローブを試料に浸漬し、3分間超音波処理する。追加の2分間は過熱から試料を冷却する。 </li><li>超音波処理した細胞溶解液をあらかじめ冷却滅菌遠心管に移し、そして二回15分間、JA-17ローター@ 35267×gで（16,000 rpm）を用いてベックマン遠心機で、4℃で遠心分離にかけられる。 注：私達は膜タンパク質である画分にわからないので、膜タンパク質の精製の ​​場合には、超音波処理した細胞溶解液を15分間遠心分離し、一度だけ、それはペレット中または上清画分のいずれかです。だからmemの過剰損失を避けるためにブレーンタンパク質は、我々は、高速遠心分離で15分間細胞をスピンさせ、上清を、その後1％の洗剤が膜タンパク質を可溶化するために使用されている場所（以下の手順を参照）精製のために処理されます。 </li><li>遠心チューブから上清を慎重に50ミリリットルポリプロピレンファルコンチューブに移し、液体窒素を用いて凍結されています。超音波処理した凍結細胞抽出物は、将来の使用のために-80℃で6ヶ月の最大期間保存されています。 </li></ol> 3。親和性精製<ol><li>使用前に、抗FLAG M2ビーズ100μlのAFCバッファー（30mMのトリス-HCl、150mMのNaCl、0.1％の洗剤、および0.1から0.5 mMのTCEP [トリス（2 -カルボキシエチル）ホスフィンの10倍量で洗浄する-洗剤なし塩酸]）と還元剤TCEP（トリス（2 - カルボキシエチル）ホスフィン）。 </li><li>凍結、超音波処理した細胞抽出物を冷たい水にチューブを配置することにより、解凍する。解凍した細胞抽出物をベンゾを3μlとインキュベートされる4℃で30分間naseヌクレアーゼこの混合物に、非イオン性界面活性剤トリトンX-100（界面活性剤の最終濃度が0.1％であるべきである）と抗FLAG M2アガロースビーズを200μlの懸濁液を追加します。 注：すべての可溶性タンパク質の精製の ​​ために、0.1％トリトンX-100などのC12E8（オクタエチレングリコールドデシルエーテル）などの膜タンパク質は、別の軽度の非イオン性界面活性剤を精製するために、DDM（非特異的吸着を最小限に抑えるために使用されn-ドデシル-β-D-マルトシド）とマルトースネオペンチルグリコール（MNG）の可溶化を高めるために、1％の界面活性剤の濃度で使用することができます。別の洗剤は、膜タンパク質の可溶化効率を変化しているので、複数の洗剤を使用して独立したタンパク質の精製を行うことを推奨します。 </li><li>コンテンツは℃LabQuake振とう機を用いて4℃で3時間真空管を回転させることにより穏やかに混合しています。回転の3時間後、チューブを6分間1700 xgで遠心分離する。上清はその後rです緩いビーズペレットを乱さずに、できるだけ、慎重にemoved。 </li><li>ペレットは、残りの上清中に再懸濁させ、その後0.8×4センチメートルBio-Rad社製ポリプロピレンプレップカラムに転送されます。溶出液は、重力流によって排水できるようにするために、カラムの底コンセントプラグを削除することを確認します。 TEV切断工程でカラムを5回洗浄します。 </li><li>洗浄された溶出液を排出した後、カラムの底部出口が閉鎖されている。同じ列に、切断は、カラムにTEVプロテアーゼおよび1X AFCのバッファーを400μlの約5から10μL（50単位）を追加することによって行われます。キャップ付きの列の上部を閉じることを確認します。ビーズを含む列がLabQuake振とう機を用いて4℃で一晩一晩静かに回転される。 </li><li>列の下部に上部とコンセントプラグのキャップを外し、10により洗浄されるカルモジュリンセファロースビーズを200μlの懸濁液を含有する新鮮な列にTEV切断後に回収した溶出液を排出カルモジュリン結合バッファーのボリューム。 </li><li>と塔の上下両方がしっかりと固定され、列の内容は℃のLabQuake振とう機を用いて4℃で3時間回転させることにより穏やかに混合する。 </li><li>回転の3時間後、溶出液をカラムの上部のキャップと底栓を除去することによって排出される。列は、カルモジュリンの洗浄バッファー400μLで1ジェントな洗浄が続く1Xカルモジュリン結合バッファー200μlで4回洗浄する。 </li><li>結合したタンパク質を1Xカルモジュリン溶出緩衝液を用いて新鮮なエッペンドルフチュー​​ブに50μlの4画分に溶出されます。溶出画分を等量の2つのきれいなエッペンドルフチュー​​ブに分配されます。両方の管が続いて高速真空を使用して乾燥させる。 </li><li>他の前の質量分析を使用し、-80℃に保存されているときに、もう1つの管からの溶出液を乾燥させ、銀染色ゲルを実行するために使用されます。 </li></ol> 4。銀染色<ol><li>銀staini用NG、3X SDS（ドデシル硫酸ナトリウム）、サンプルバッファの半分のボリュームは、乾燥した溶出液の50μlに追加されます。 5分間煮沸した後、試料をSDSポリアクリルアミドゲル上にロードされます。 </li><li>ゲルの実行が終了した後、それを慎重に固定液に入れられ（50％メタノールと10％酢酸）と20分間ロータリーシェーカーで穏やかに攪拌されています。その後、ゲルを10分間、20％エタノールでリンスする。 </li><li>固定されたゲルは低い均一な背景を確実にするために10分間再蒸留水500mlで2回徹底的に洗浄する。 </li><li>その後、ゲルを20秒間、蒸留水で2回の洗浄工程に続いて1分間、500mlのチオ硫酸ナトリウムで穏やかに攪拌されています。 </li><li>水を捨て、ゲルを30分間0.1％硝酸銀の200ミリリットルでインキュベートする。その時間が経過した後、硝酸銀が削除され、ゲルは、過剰の硝酸銀を削除するには、20秒間、蒸留水で洗浄する。 </li><li>ゲルがついに75mlに撹拌手である現像液の。所望の強度が達成されると、現像液は廃棄され、酢酸80mlを加えて反応を停止しています。ゲルのバンドの適切な可視化のための20分間の最小値に対して酢酸でゲルをインキュベートすることを確認します。 </li></ol> 5。質量分析のためのタンパク質分解とサンプルの調製<ol><li>乾燥した試料に、消化緩衝液50μlおよび100mM TCEP-塩酸（トリス（2 -カルボキシエチル）ホスフィン-塩酸）0.9μlを添加し、室温で45分間混合物をインキュベート 還元工程のために。次に、500mMのヨードアセトアミドを1μl添加し、サンプルのアルキル化を可能にするために別の40分間、暗所でインキュベートする。 </li><li>インキュベーションの第二ラウンド後、この混合物に固定化トリプシンの1μgを追加し、5時間または室温で一晩37℃でインキュベートどちらか。酢酸1μlを加えて反応を停止することを確認します。 </li><li>プリウェットMillipo湿潤および平衡溶液10μlを吸引による再ジップヒントピペットチップ。無駄にするソリューションを免ずる。その後、洗浄溶液10μLを吸引し、無駄にするソリューションを免ずる。二回、この手順を繰り返します。 </li><li>先端までのペプチド混合物の効率的な結合は、ピペットは、ペプチド混合物を20回混ぜる。先端に付着したペプチド混合物を吸引し、洗浄溶液を分注することにより洗い流される。先端までのペプチド混合物の効率的な結合のために二度、この手順を繰り返します。 </li><li>結合したペプチドを含む先端で、湿潤および平衡溶液10μlを吸引し、クリーンなエッペンドルフチュー​​ブに分注する。この工程を2回繰り返します。使用する°Cの前速度、真空中で溶出した試料を乾燥させた後、試料を質量分析によって直ちに解析することができ、または-80℃で保存。 </li></ol> 6。 LTQオービトラップVelos質量分析計によるタンパク質同定木曜日隔離された複合体の電子ポリペプチド成分はのLTQオービトラップVelosハイブリッドタンデム質量分析計を使用して識別されます。オービトラップは高速Velosイオントラップしながら、コントロール精製で検出無関係な非特異的なバックグラウンド汚染物のMS / MSサンプリングを最小限に抑えることが非常に優れた解像力（&gt; 60,000全幅半値か、FWHM）と質量精度（&lt;2 ppm）を持ってコンポーネントは、電子移動解離と衝突誘起解離の両方のモードを使用して、低濃度のペプチドを検出およびフラグメントすることができます。結果としてMS / MSスペクトルの間で高い信頼マッチはリファレンスEにマップされSEQUESTとSTATQUEST 11のような確率アルゴリズムの実行中は、評価した各照合シーケンスなどのデータベース検索アルゴリズムを使用して大腸菌タンパク質配列。合計スペクトル番号、陰性コントロール（ すなわち 。モック）精製で検出されたペプチド配列の一意性と共通のバックグラウンド汚染物質をfalse-DISロー経験を達成するために考えられているcovery率。次の手順は、タンパク質の同定のために実行されています： <ol><li>マイクロカラムが3μmルナ〜C 18樹脂の約10 cmの満員とオービトラップ器に沿って配置されProxeonナノエレクトロスプレーイオン源にインタフェースされています。 </li><li> ProxeonナノフローバイナリHPLCポンプは、ペプチド分離の間に約300 nlの分-1の安定した先端流量を提供する目的で使用されます。 </li><li>ペプチド溶出を達成するために、有機緩衝勾配は、サンプルの複雑さに応じて設定されています。たとえば、次のような勾配は、通常、 大腸菌用に設定されている大腸菌 SPA のサンプル：溶媒Bは1分間100パーセントで開催された次の4分間、100％に、38分で24％に、1分間に2〜6％から増加しているが、その後、1分で2％に減少そして最終的には15分間2％に保持します。溶媒Bが5％、HPLCグラジエント水および0.1％ギACIと95％ACNを有しているが、0.1％のギ酸と95％HPLCの勾配の水と5％ACNを持って移動相溶媒D。針の先端で流量を60分間300 nlの分-1に設定されます。 </li><li>質量分析計サイクルが60,000の解像度でフル1質量走査を介して実行すると、10併用タンデムフラグメント化質量スキャンが最も強烈な前駆体イオンのために収集されています。動的除外、モノアイソトピック質量の選択、およびリアルタイムデータ依存取得装置機能が有効になっています。 </li><li>スペクトルは、例えば大腸菌のデータベースに対するSEQUESTなどのデータベース検索アルゴリズムを用いて検索されます大腸菌のタンパク質配列し、その結果を統計的に低い偽の発見率を確保するためにそのようなSTAQUEST 11として確率アルゴリズムを使用してフィルタリングされます。マッチが理論的なペプチド質量に比べて10 ppmの質量誤差を示すさらなる検討の対象から除かれている間のみ、高い信頼性（99％確率）候補が、選択されています。 </li></ol>

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	<li>Butland, G., 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=Interaction+network+containing+conserved+and+essential+protein+complexes+in+Escherichia+coli.">Interaction network containing conserved and essential protein complexes in Escherichia coli.</a> Nature. 433, 531-537 (2005).</li><li>Hu, P., Janga, S. C., Babu, M., Diaz-Mejia, J. J., Butland, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+functional+atlas+of+Escherichia+coli+encompassing+previously+uncharacterized+proteins.">Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins.</a> PLoS Biol. 7, e1000096 (2009).</li><li>Krogan, N. J., 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=Global+landscape+of+protein+complexes+in+the+yeast+Saccharomyces+cerevisiae.">Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.</a> Nature. 440, 637-643 (2006).</li><li>Mak, A. B., 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=A+lentiviral+functional+proteomics+approach+identifies+chromatin+remodeling+complexes+important+for+the+induction+of+pluripotency.">A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency.</a> Mol. Cell Proteomics. 9, 811-823 (2010).</li><li>Polanowska, J., 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=Tandem+immunoaffinity+purification+of+protein+complexes+from+Caenorhabditis+elegans.">Tandem immunoaffinity purification of protein complexes from Caenorhabditis elegans.</a> Biotechniques. 36, 778-782 (2004).</li><li>Veraksa, A., Bauer, A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein+complexes+in+Drosophila+with+tandem+affinity+purification-mass+spectrometry.">Analyzing protein complexes in Drosophila with tandem affinity purification-mass spectrometry.</a> Dev. Dyn. 232, 827-834 (2005).</li><li>Zeghouf, M., 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=Sequential+Peptide+Affinity+(SPA)+system+for+the+identification+of+mammalian+and+bacterial+protein+complexes.">Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes.</a> J. Proteome Res. 3, 463-468 (2004).</li><li>Puig, O., 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=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Rigaut, G., 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=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat. Biotechnol. 17, 1030-1032 (1999).</li><li>Yu, D., 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=An+efficient+recombination+system+for+chromosome+engineering+in+Escherichia+coli.">An efficient recombination system for chromosome engineering in Escherichia coli.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 5978-5983 (2000).</li><li>Kislinger, T., 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=PRISM,+a+generic+large+scale+proteomic+investigation+strategy+for+mammals.">PRISM, a generic large scale proteomic investigation strategy for mammals.</a> Mol. Cell Proteomics. 2, 96-106 (2003).</li><li>Vlasblom, J., 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=GenePro:+a+Cytoscape+plug-in+for+advanced+visualization+and+analysis+of+interaction+networks.">GenePro: a Cytoscape plug-in for advanced visualization and analysis of interaction networks.</a> Bioinformatics. 22, 2178-219 (2006).</li><li>de Berardinis, V., 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=A+complete+collection+of+single-gene+deletion+mutants+of+Acinetobacter+baylyi+ADP1.">A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1.</a> Mol. Syst. Biol. 4, 174 (2008).</li><li>Babu, M., 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=Sequential+peptide+affinity+purification+system+for+the+systematic+isolation+and+identification+of+protein+complexes+from+Escherichia+coli.">Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.</a> Methods Mol. Biol. 564, 373-400 (2009).</li></ol>

特別な利害関係は宣言されません。

ここで説明するSPAベースAPMSアプローチの重要な側面は、タギングがそれによって正常な遺伝子調節を確保し、自然な染色コンテキスト内で実行される維持（ すなわち 。ネイティブ餌プロモーターが保持され、それ故に発現レベルが乱されていない）と安定的に結合したネイティブであるということですタンパク質複合体は、近内在性レベルで回収される。オペロンの極性の問題は、?...

<table border="1">
	<tbody>
		<tr>
			<td>Materials</td>
			<td>Vendor</td>
			<td>and Catalog Numbers</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>I. Antibiotics</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Bioshop</td>
			<td>#KAN201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>#AMP201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Terrific-Broth medium</td>
		</tr>
		<tr>
			<td>Bio-Tryptone</td>
			<td>Bioshop</td>
			<td>#TRP 402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bioshop</td>
			<td>#YEX 555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Bioshop</td>
			<td>#GLY 002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Bioshop</td>
			<td>#PPM 302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Bioshop</td>
			<td>#PPM 303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. Bacterial Strain and Plasmid</td>
		</tr>
		<tr>
			<td>DY330</td>
			<td>&nbsp;</td>
			<td>Yu et al. (2000)10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pJL148</td>
			<td>&nbsp;</td>
			<td>Zeghouf et al. (2004)7</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. PCR and Electrophoresis Reagents</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 X PCR buffer</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10 mM dNTPs</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 mM MgCl2</td>
			<td>Fermentas</td>
			<td># EP0281</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td># AGA002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Loading dye</td>
			<td>NEB</td>
			<td>#B7021S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Bioshop</td>
			<td># ETB444</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Thermo Scientific</td>
			<td>#28355</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Bioshop</td>
			<td>#TRS001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Bioshop</td>
			<td>#BOR001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Sigma</td>
			<td># E6768</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNA ladder</td>
			<td>NEB</td>
			<td>#N3232L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. Plasmid isolation and Clean-up Kits</td>
		</tr>
		<tr>
			<td>Plasmid Midi kit</td>
			<td>Qiagen</td>
			<td>#12143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>#28104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. PCR and Transformation Equipments</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>BioRad</td>
			<td>iCycler</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis</td>
			<td>BioRad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-Rad</td>
			<td>GenePulser II</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.2 cm electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>42 &deg;C water bath shaker</td>
			<td>&nbsp;</td>
			<td>Innova 3100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Beckman Coulter TJ-25 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>TS-5.1-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C Shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C large Shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>32 &deg;C plate incubator</td>
			<td>Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>7. Electrophoresis and Western blotting</td>
		</tr>
		<tr>
			<td>Acrylamide monomer, N,N'- methylenebis-acrylamide</td>
			<td>Bio-Rad</td>
			<td>#161-0125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Bioshop</td>
			<td># AMP001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>n-butanol</td>
			<td>Sigma</td>
			<td># B7906</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bioshop</td>
			<td>#TEM001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Whatman No. 1 filter paper</td>
			<td>Fischer Scientific</td>
			<td>#09-806A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mini protean 3 cell</td>
			<td>Bio-Rad</td>
			<td>#165-3301</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>iBlot gel transfer device</td>
			<td>Invitrogen</td>
			<td>#IB1001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>Bio-Rad</td>
			<td>#162-0115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Flag M2 antibody</td>
			<td>Sigma</td>
			<td>#F3165</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Amersham</td>
			<td>#NA931V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pre-stained protein molecular weight standards</td>
			<td>Bio-Rad</td>
			<td>#161-0363</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chemiluminescence reagent</td>
			<td>PIERCE</td>
			<td>#1856136</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Autoradiography film</td>
			<td>Clonex Corp</td>
			<td>#CLEC810</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Quick Draw blotting paper</td>
			<td>Sigma</td>
			<td>#P7796</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C2 platform rocking shaker</td>
			<td>New Brunswick Scientific, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>8. Sonication Equipment and Reagents</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Branson Ultrasonic</td>
			<td>#23395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>#SOD001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche</td>
			<td>#800-363-5887</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.5 mM TCEP-HCl</td>
			<td>Thermo Scientific</td>
			<td>#20490</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>9. Affinity Purification Reagents and Equipment</td>
		</tr>
		<tr>
			<td>0.8 x 4 cm Bio-Rad polypropylene column</td>
			<td>Bio-Rad</td>
			<td>#732-6008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzonase nuclease</td>
			<td>Novagen</td>
			<td>#70746</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-FLAG M2 agarose beads</td>
			<td>Sigma</td>
			<td>#A2220</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calmodulin-sepharose beads</td>
			<td>GE Healthcare</td>
			<td>#17-0529-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TEV protease</td>
			<td>Invitrogen</td>
			<td>#12575-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>#T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>#C2661</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>#E3889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LabQuake Shaker</td>
			<td>Thermolyne</td>
			<td>#59558</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>10. Silver Staining Reagents</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Bioshop</td>
			<td>#MET302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Bioshop</td>
			<td>t#ACE222</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium-thiosulfate</td>
			<td>Sigma</td>
			<td>#S-7143</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Fischer Scientific</td>
			<td>#S181-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Bioshop</td>
			<td>#FOR201</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Bioshop</td>
			<td>#SOC512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>11. Reagents and Equipment for Protein Identification </td>
		</tr>
		<tr>
			<td>Trypsin Gold, Mass Spectrometry Grade</td>
			<td>Promega</td>
			<td># V5280</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 mM NH4HCO3</td>
			<td>Bioshop</td>
			<td>#AMC555</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 mM CaCl2</td>
			<td>Bioshop</td>
			<td>#CCL302</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma</td>
			<td>#A998-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma</td>
			<td>#F0507</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>Sigma</td>
			<td>#95304</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma</td>
			<td>#16125</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Millipore Zip-Tip</td>
			<td>Millipore</td>
			<td># ZTC18M960</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>~10 cm of 3 &mu;m Luna-C18 resin</td>
			<td>Phenomenex</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Proxeon nano HPLC pump</td>
			<td>Thermo Fisher Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos mass spectrometer</td>
			<td>&nbsp;</td>
			<td>Thermo Fisher Scientific</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>12. Labware</td>
		</tr>
		<tr>
			<td>4 liter conical flasks</td>
			<td>VWR</td>
			<td>#89000-372</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml polypropylene falcon tubes</td>
			<td>Any Vendor</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.5 ml micro-centrifuge tubes</td>
			<td>Any Vendor</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>250 ml conical flaks</td>
			<td>VWR</td>
			<td>#29140-045</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml sterile culture tubes</td>
			<td>Thermo Scientific</td>
			<td>#366052</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>VWR</td>
			<td>#479-3221</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>-80 &deg;C freezer</td>
			<td>Fisher Scientific</td>
			<td>#13-990-14</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Speed vacuum system</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Buffers and Solutions
			1. 1 liter Terrific Broth (TB) media
			11 g Bio-Tryptone 
			22 g Yeast Extract 
			2% Glycerol 
			50 ml potassium salt stock solution
			2. Potassium Salt Stock Solution
			1.5 M K2HPO4 
			0.35 M KH2PO4
			3. Sonication Buffer
			20 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.2 mM EDTA 
			10% Glycerol 
			Before use add protease inhibitor (PI) and 0.1-0.5 mM TCEP
			4. AFC buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			5. TEV cleavage buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			0.2 mM EDTA 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			6. Calmodulin binding buffer
			30 mM Tris-HCl (pH 7.9) 
			150 mM NaCl 
			2 mM CaCl2 
			0.1% detergent 
			Before use add PI and 0.1-0.5 mM TCEP
			7. Calmodulin wash buffer
			30 mM Tris-HCl pH 7.9 
			150 mM NaCl 
			2 mM CaCl2 
			0.1-0.5 mM TCEP
			8. Calmodulin elution buffer
			30 mM Tris-HCl (pH 7.9) 
			100 mM NaCl 
			10 mM EGTA 
			0.1-0.5 mM TCEP
			9. Developing solution (1L)
			37% Formaldehyde 
			30 g sodium carbonate 
			1000 ml distilled water
			10. Digestion buffer
			50 mM NH4HCO3 
			1 mM CaCl2
			11. Wetting and Equilibration solution
			70% acetonitrile (ACN) in 0.1% formic acid
			12. Washing solution
			100% H2O in 0.1% formic acid</td>
		</tr>
	</tbody>
</table>

identification of protein complexes in escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry

ほとんどの細胞プロセスを高分子アセンブリによって媒介されているので、タンパク質-タンパク質相互作用（PPI）および多タンパク質複合体のサブユニット組成の同定を体系的に同定は、遺伝子の機能への洞察を提供し、生物学的システム1,2の理解を高めることができます。物理的な相互作用は、質量分析法（APMS）と組み合わせて染色体タグ融合タンパク質のアフィニティー精製に基づいて近い生理的条件下で内因性のタンパク質複合体の高い信頼vialargeスケールの単離および特性をマッピングすることができます。このアプローチは正常酵母、ハエ、虫、哺乳類細胞、細菌1月6日を含め進化的に多様な生物に適用されています。特に、我々はGEを使用して、培養したグラム陰性大腸菌からアフィニティー精製、天然のタンパク質複合体のためのカルボキシ末端シーケンシャルペプチドアフィニティー（SPA）をデュアルタギングシステムを生成した基礎生物学と生物1、2、7の保存プロセスのゲノムワイドな研究に適していnetically-扱いやすいホスト実験室株。当社SPA-タグシステムは、酵母8,9のために最初に開発されたタンデムアフィニティー精製法に類似していて、非常に特異的なタバコエッチウイルス （TEV）プロテアーゼと3つのコピーの切断部位が続くカルモジュリン結合ペプチド（CBP）から構成されていFLAGエピトープ（3X FLAG）の、親和性濃縮の2連続ラウンドを可能にする。カセットの増幅後、SPA-タグおよび選択可能なマーカーをコードする配列特異線形PCR産物が統合されており、一時的に非常に効率的な異種バクテリオファージラムダ組換え系10の発現が誘導さDY330背景にカルボキシ末端融合としてフレームで表現されます。カルモジュリンと抗FLAGアフィニティービーズを使用して、後続のデュアルステップ精製が高度に選択が可能大規模な文化からでも低濃度のタンパク質複合体と効率的な回収。タンデム質量分析を高感度（低ナノグラム検出限界）で安定して共同精製タンパク質を同定するために使用されます。 ここでは、 大腸菌由来の可溶性タンパク質複合体の精製と質量分析ベースの分析、我々は一般的に体系的タンパク質のタグ付けに使用する詳細なステップバイステップの手順を説明します大腸菌 、スケールアップと潜在的にコンビに従順である特定の日和見病原体を含む他の細菌種に合わせて調整することができます。結果の物理的相互作用は、しばしば小説序のリンクを示唆して興味深い予想外のコンポーネントと接続を明らかにすることができます。予測はBI内多タンパク質複合体のグローバルな分子組織の解明を容易にすることができますこのような遺伝的（遺伝子遺伝子）の相互作用とゲノム·コンテキスト（GC）などの代替分子会合データとPPIデータの統合ological経路。 大腸菌に対して生成されたネットワーク大腸菌は、機能アノテーションは現在欠けているために他の微生物のオーソログ遺伝子産物の機能アーキテクチャへの洞察を得るために使用することができます。

Once tagged bait proteins, which are expressed at endogenous levels are affinity-purified from logarithmic phase cultures the samples were run on a silver-stain gel to visualize the individual polypeptide components of the isolated stable complexes. We also subjected a second portion of the affinity-purified protein samples to gel-free tandem mass spectrometry (LCMS) to identify the corresponding polypeptide sequences. The effectiveness of this APMS procedure is shown with a representative SDS-PAGE analysis of the compon...

Watch this Scientific Journal Video about Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry at JoVE.co

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

マススペクトロメトリー（APMS）との組み合わせでタグ付けされたタンパク質のアフィニティー精製は、タンパク質相互作用ネットワークの体系的マッピングのため、生物学的プロセスの基本メカニズムを調査するための強力な方法です。ここでは、細菌のために開発最適化された順次ペプチドアフィニティー（SPA）をAPMSの手順を説明<em&gt;大腸菌</emでも、セルあたりの低コピー数から始まるほぼ均一に安定した多タンパク質複合体を分離し、特徴付けるために使用することができます&gt;。

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

identification-protein-complexes-escherichia-coli-using-sequential

Research

JoVE Journal

Biology

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

48.2K ビュー.  University of Toronto. マススペクトロメトリー（APMS）との組み合わせでタグ付けされたタンパク質のアフィニティー精製は、タンパク質相互作用ネットワークの体系的マッピングのため、生物学的プロセスの基本メカニズムを調査するための強力な方法です。ここでは、細菌のために開発最適化された順次ペプチドアフィニティー（SPA）をAPMSの手順を説明大腸菌

ビデオ: Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

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 ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

タンデムアフィニティー精製を用いたタンパク質相互作用するパートナーの識別

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

に基づいて、タンパク質 - タンパク質相互作用の同定のためのプロトコル<sup&gt; 15</sup&gt; Nの代謝標識、免疫沈降法、定量的質量分析法と親和性調節

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

中のタンパク質 - タンパク質相互作用を特定する<em&gt;ショウジョウバエ</emタンデムアフィニティー精製（TAP）による&gt;アダルトヘッド

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

からのペプチドおよびタンパク質との組換えエラスチン様ポリペプチドおよびその融合の非クロマトグラフィー精製<em&gt;エシェリヒア·コリ</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

タンパク質の共発現の多面的メリットで<em&gt;エシェリヒア·コリ</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...