This work was funded by the Wellcome Trust (WT098051).

<ol>
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The authors have nothing to disclose.

ここでは、タンパク質複合体を解決するために、定量的質量分析と組み合わせたブルーネイティブゲル電気泳動、続いてアフィニティー精製の使用を記載しています。このアプローチは、機能性タンパク質アセンブリに1次元のタンパク質相互作用のリストを解明するための方法を提供しています。 私たちは、エピトープタグ融合タンパク質の使用に基づく方法を示しています。しかし、タグ付けされたタンパク質を発現する細胞株が利用できない場合、代替は、目的のタンパク質に対する抗体を使用するのが良いかもしれない、ネイティブの競合溶出を達成することが可能なペプチドが提供されます。出発材料とビーズの量の量は、標的タンパク質の発現レベルに応じて変更する必要があるかもしれません。我々は、典型的には、出発材料2-5×10 8細胞を用いて、このプロトコルを実行し、これも低い発現レベルを有するタンパク質に十分です。溶解緩衝液の組成は、達成するために経験的に選択されるべきです近くに餌の可溶化を完了します。これは、特定のクロマチン結合または膜タンパク質で、タンパク質のいくつかのタイプのために挑戦される可能性があります。代替オプションは、研究下のタンパク質複合体は、高塩、またはクロマチン結合タンパク質20、29のための超音波処理および/またはヌクレアーゼ処理を使用して安定しているという条件で、塩の量を増加させることを含みます。膜タンパク質の場合には、DDMまたはジギトニンに洗剤を切り替えは30が望ましいことがあります。 DNA結合タンパク質の場合、精製工程20中にこのようなベンゾナーゼヌクレアーゼを含むことが有用です。核酸の完全な除去は、検出された相互作用は、タンパク質間で発生し、DNAによって媒介されないことを保証します。 このアプローチを使用する際に考慮すべき重要な要素は、調査中の複合体の安定性です。手順が長く、preservinを含むことができますGタンパク質複合体で一晩。我々は2つのクロマチンリモデリング複合体（D.ボーデとM.パルド、データは示していない）との良好な成功を収めているが、これは評価されるべきです。必要に応じてアフィニティー精製工程を短縮することができます。 サイズ排除クロマトグラフィーなどのネイティブの分別を許可別の技術は、広くタンパク質複合体を特徴づけるために50年以上にわたって使用されています。我々および他のものはブルーネイティブPAGEの解像度は、サイズ排除クロマトグラフィー20、31、32、33を用いて達成されるものよりも優れていることを示しています。ブルーネイティブPAGEのもう一つの利点は、それが高価なクロマトグラフィーシステムを、必要としないことであるが、むしろ研究室で広く普及しているタンパク質の電気泳動装置を使用しています。ハンズオン時間の面では、この方法では、伝統的なGELC-MS / MS Aより多くの作業を必要としませんpproachまたはオフラインのクロマトグラフィー分画。ほとんどの分別技術がそうであるようにしかし、それは彼らの解像度に制限があります。質量および形状が非常に均一または近接している複合体は、青色ネイティブPAGEによって提供される解像度を超えていてもよく、従ってここで報告されているように、プロトコルは、共有サブユニットと別個の複合体を解決するために普遍的に成功しないかもしれません。心強いことに、我々は3つのサブユニット（M.パルド、投稿準備中）を共有する2つの非常に類似した四量体複合体を分離することに成功しています。 質量分析はますます敏感になってきているので、非特異的相互作用と汚染物も微量ではAP-MSサンプルにおいて検出することができます。ここで紹介する方法は、より高い分子量の餌移行ピークと一致する移行ピークを有するタンパク質に着目してアフィニティ精製におけるバックグラウンド汚染から実際の相互作用の識別に役立つ可能性があります単量体タンパク質。 いくつかのグループは、前の分離10、11、12、34を必要とせずに細胞スケールでのタンパク質複合体を描写するタンパク質相関プロファイリング続く分別技術を使用しています。しかしながら、これは、準化学量論的相互作用を検出するための故障をもたらすことができます。アフィニティ精製によって濃縮ステップの取り込みは、これを克服するのを助けることができます。ここで説明するアプローチは、タンパク質複合体のトポロジーを探索し、所定のタンパク質が同じ細胞コンテキスト内に参加する複数の複合体を解明するための一般的に有用であるべきです。戦略は、タンパク質複合体を解決するために、高価なクロマトグラフィー分画の機器を持っていない可能性が研究室にシンプルかつ従順です。

細胞では、ほとんどのタンパク質は、一過性のタンパク質 - タンパク質相互作用を介して、または安定なタンパク質の集合体を形成することにより、その機能を実行します。タンパク質相互作用を特徴づけることは十分に理解細胞プロセスのために重要です。質量分析法（AP-MS）と組み合わせたアフィニティー精製は、ネイティブのタンパク質相互作用を同定するための最も一般的に適用される戦略の一つです。過去十年間で達成機器の機能の大幅な改善は、このアプローチは非常に強力作られてきました。 AP-MS実験によって識別される相互作用が餌と獲物の間の直接および間接的な団体の混合物が含まれることに注意することが重要です。加えて、多くの場合、タンパク質は異なる生物学的役割を有するかもしれない同じ細胞コンテキスト内でいくつかの異なる複合体に関与し、したがってAP-MSによって同定された相互作用は、異なるタンパク質集合体または機能エンティティの組み合わせを表すかもしれません。導出することはできませんそのような位相情報単純なAP-MS実験によって生成されたタンパク質の一次元リストから演繹 。しかし、この技術は、これらのアセンブリを解決するために、1つまたは複数の方法と組み合わせることにより、タンパク質複合体の構造を定義するためにさらに利用することができます。 AP-MSによって同定されたタンパク質間相互作用のトポロジーを解決するために、いくつかの戦略が適用されています。一つのアプローチは、餌1と実験の前のラウンドで同定された獲物を用いて反復AP-MS実験を実行することです。非常に有益が、これは両方の実験的および解析的に非常に労働集約的な作業です。質量分析と組み合わせたタンパク質架橋は、ますますタンパク質複合体2、3、4、5で位相情報を導出するために使用されています。しかし、computatio架橋されたペプチドの最終分析は、依然として困難な作業のままであり、したがって、ワークフローのボトルネックです。 MS-開裂可能な架橋試薬の出現は、タンパク質6、7相互作用に近接しているアミノ酸残基のマッピングを容易にするはずです。別の代替は、従来の直交分離技術8,9との親和性精製を組み合わせることです。ゲル濾過またはイオン交換によるクロマトグラフィー分画、またはスクロース勾配分画はまた、最近、通過複合分離工程10、11、12、13、システム全体のレベルでの多タンパク質複合体を記述するために、定量的質量分析と組み合わせて使用されてきました。ブルーネイティブポリアクリルアミドゲル電気泳動（BN-PAGE）が広くに適用されています天然のタンパク質の相互作用、ミトコンドリア膜タンパク質複合体14を含む一般的に、これらを調査します。この分離技術はまた、最近、ミトコンドリア複合体15、16、17上だけでなく、全体のセル18、19からの他のタンパク質複合体を解明するためだけでなく、ラベルフリータンパク質定量と相関プロファイリングと組み合わせて使用しました。我々は、その後のネイティブ分画手法および定量的MSとの親和性精製の組み合わせは、特定の蛋白質を含む複数タンパク質アセンブリを解決するための有用な戦略を提供するはずであるという仮説を立てました。 ここでは、定量的質量、SP、続いて単離された複合体のブルーネイティブポリアクリルアミドゲル電気泳動を用いて、一般的なエピトープベースのアフィニティー精製を組み合わせた方法を記載しectrometryとタンパク質相関プロファイリングは、タンパク質が関与する可能性がある複数のアセンブリを解決することができます。私たちは、エピトープタグに融合した目的のタンパク質が生理的豊かさに近い達成し、効率的なネイティブを確保するために、内因性遺伝子座から発現されたマウス胚性幹細胞を使用します複雑な分離。このアプローチは、複数の相互作用タンパク質は、従来GELC-MS 20よりも多くの作業を必要としない一方で、それらの相関プロファイルに基づいて異なるアセンブリにそれらを解決する、に係合を解きます。

<table border="0" cellpadding="0" cellspacing="0" width="1384">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Protein G Dynabeads</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">10003D</td>
			<td style="width:452px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">FLAG antibody M2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Tween-20, protein grade, 10% solution</td>
			<td style="width:285px;">Millipore</td>
			<td style="width:220px;">655206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Dimethyl pimelimidate</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">80490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">0.2 M Triethanolamine buffer, pH 8.2</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">T0449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">3x FLAG peptide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">F4799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Vivaspin 5K NMWCO, PES</td>
			<td style="width:285px;">Sartorius</td>
			<td style="width:220px;">VS0112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativePAGE 3-12% Bis-Tris gel</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Running Buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">20x NativePAGE Cathode Additive</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">4x NativePAGE sample loading buffer</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">NativeMARK</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">LC0725</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NativePAGE 5% G-250 sample additive&#160;</td>
			<td style="width:285px;">Life Technologies</td>
			<td style="width:220px;">BN2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc conical bottom 96-well plate, polypropylene</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">249944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Multiscreen HTS 96-well filtration system</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">MSDVN6550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Nunc 96-well cap natural</td>
			<td style="width:285px;">Thermo</td>
			<td style="width:220px;">276002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">TCEP (Tris(2-carboxyethyl)phosphine hydrochloride)</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">646547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Iodoacetamide</td>
			<td style="width:285px;">Sigma-Aldrich</td>
			<td style="width:220px;">I1149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Acetonitrile, HPLC grade</td>
			<td style="width:285px;">Fisher Scientific</td>
			<td style="width:220px;">A/0627/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Trypsin, sequencing grade</td>
			<td style="width:285px;">Roche</td>
			<td style="width:220px;">11418475001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Software</td>
			<td style="width:285px;"></td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">MaxQuant (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111795/maxquant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Perseus (software)</td>
			<td style="width:285px;">N.A.</td>
			<td style="width:220px;">N.A.</td>
			<td style="width:452px;">http://www.biochem.mpg.de/5111810/perseus</td>
		</tr>
	</tbody>
</table>

resolving affinity purified protein complexes by blue native page and protein correlation profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

質量分析によるアフィニティー精製ブルー天然タンパク質相関プロファイリングのワークフローは、（ABC-MS）戦略が、図7に示されています。関心対象のタンパク質の周り天然タンパク質複合体（この場合、フラグに）エピトープタグに対する抗体を用いたアフィニティー精製および競合的溶出によって単離されます。複合体は、BN-PAGEによって分離され、そして全体のゲルレーンを48個のセクションに切り出し、ショットガンLC-MS / MSのために準備されます。定量的MS情報は、青色ネイティブ分離挟んで同定されたタンパク質の移行プロファイルを生成するために使用されます。重なり合うピークを有する別個の複合表示同様の移動プロファイルを形成するように相互作用するタンパク質。関心対象のタンパク質が複数のアセンブリに関与する場合、複数のピークは、その移行プロファイルで観察されたサブ複合体は、ブルーネイティブゲルの分解能の範囲内である与えられています。 MIGの体系的な比較配給プロファイルは、階層的クラスタリングを使用してプロファイリングタンパク質相関によって達成することができます。すべての画分についてのデンドログラムとピーク強度は、異なるタンパク質複合体（ 図8）に属するタンパク質相互作用の同定を容易に、ヒートマップに可視化されます。 我々はMTA2、複雑な20を改造NURDクロマチンのコアサブユニットの相互作用パートナーを分析するために、この戦略を使用していました。 図9に示すように、MTA2の移動プロファイルは、より高い存在量を表示低い質量ピークと、700 kDaの1.2 MDA間の異なる強度の2つのピークを示しました。 MTA1 / 3、HDAC1 / 2とMBD3を含む他のNURDコアサブユニットは、サブユニット、すなわちChd4の一部についてのピークはあるが、同一の分離パターンを示し、B Gatad2a /及びRbbp4 / 7は、（図9A逆存在量分布を有しB）。これとは対照的に、のCdkのプロフィール2ap1は、Wnt遺伝子のプロモーター23にNURD複合体を動員調節因子は、唯一のより高い質量ピーク（ 図9C）を表示し、そうSALL4もNURD 20、24、25結合することが示されている転写リプレッサーを行いました。したがって、ブルーネイティブPAGEによってアフィニティー精製MTA2関連タンパク質の分画は、NURD複合体の2つの異なる形式を解決することができました。 ABC-MSはまた、特定のタンパク質のエンティティに割り当てる一方、新規相互作用の同定を可能にします。ヒートマップで表されるタンパク質クラスターと分数強度の検査を通じて、我々はWDR5 2つのNURD Pと一致する2つの移行のピークを示すと共に、NURDサブユニットとWDR5、MLLメチルトランスフェラーゼ複合体26の調節サブユニットとの間の強い相関を検出しましたWDR5とNURDの間の新規の相互作用を示唆しeaks（ 図8）。私たちは、共免疫沈降およびサイズ排除クロマトグラフィー20での同時移行により、この相互作用を確認しました。 階層的クラスタリングにおける距離メトリック計算の選択は、クラスタひいては相関の形状に影響を与えるであろう。私たちは、目的のタンパク質または複合体の既存の相互作用の知識を持つ最高のフィット感を実現するために異なるメトリックを試してお勧めします。一般的に、我々は、マンハッタン（L1）距離メトリック20で最良の結果を達成しました。ピアソン相関やユークリッド距離メトリックは、代替分別技術10、11、12に基づいて、複合体を同定するために報告されています。 泉ブルーネイティブ画分から得られたtitative MSデータはまた、タンパク質複合体の化学量論を決定するために用いることができます。 iBAQ（強度基づいて絶対定量）値は、同定されたタンパク質27、28の相対的存在量の尺度を提供します。マイグレーションプロファイルピーク内の全ての画分中のタンパク質複合体メンバーのiBAQ値は、相対的な量を得るために、ベイトタンパク質のそれに正規化されます。所与のタンパク質複合体のプロファイルのピークを横切る正規iBAQs水平トレンドに従うべきであり、トレンド値は、ベイトタンパク質に対する相互作用タンパク質の化学量論を反映します。化学量論計算のより詳細な表現については、20を参照してください。 <img alt="図7" src="/files/ftp_upload/55498/55498fig7.jpg" /> 図7：ABC-MS Sを要約する概略ワークフローtrategy。この図は、20から変更されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig7large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図8" src="/files/ftp_upload/55498/55498fig8.jpg" /> MTA2の相互作用のBN-PAGE移動プロファイルの図8の階層的クラスタリング。 MTA2関連タンパク質は、それらの移動プロファイルの類似性に基づいてクラスタ化されました。 NURD錯体を含有するヒートマップの一部のみが（青色のボックスに囲まれている）に示されています。黄色のボックスは、NURD複合体とWDR5の強い相関関係を強調しています。注釈付きの分子量は、同一ゲルで実行タンパク質標準の移動距離から推定しました。この研究は、もともと生化学とMoのためのアメリカの社会は、分子細胞プロテオミクス20に掲載されました<html> lecular生物学。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/55498fig8large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図9" src="/files/ftp_upload/55498/55498fig9.jpg" /> NURDサブユニットおよび関連タンパク質の図9 BN-PAGEの移行プロファイル。 A）同様の強度パターンに存在MTA2とNURDサブユニット。逆強度パターンを有するB）MTA2とNURDサブユニット。唯一のより高い分子量のNURDエンティティ中に存在するC）MTA2とCdk2ap1、。注釈付きの分子量は、タンパク質標準の移動プロファイルから推定しました。この研究は、もともと分子細胞プロテオミクス20生化学・分子生物学のためのアメリカの社会に掲載されました。_blank &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="補足ファイル" src="/files/ftp_upload/55498/proteinGroupsDB04.jpg" /> 補足データ：サンプルデータセット。 ABC-MS実験からのタンパク質同定および定量値を含むMaxQuant由来proteingroups.txtファイル。この研究は、もともと分子細胞プロテオミクス20生化学・分子生物学のためのアメリカの社会に掲載されました。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55498/proteinGroupsDB04.txt" target="_blank">このファイルをダウンロードするにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling at JoVE.com

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

ここでは、ラベルフリーの定量的質量分析法を用いたタンパク質相関プロファイリングに続く親和性タンパク質複合体の精製と青ネイティブPAGEによるそれらの分離のためのプロトコルを提示します。この方法は、異なるタンパク質複合体へのインタラクトームを解決するのに便利です。

ブルーネイティブPAGEおよびタンパク質相関プロファイリングによってアフィニティー精製タンパク質複合体の解決

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

resolving-affinity-purified-protein-complexes-blue-native-page

Nanyang Technological University

Research

JoVE Journal

Biochemistry

13.7K ビュー.  Wellcome Trust Sanger Institute. ここでは、ラベルフリーの定量的質量分析法を用いたタンパク質相関プロファイリングに続く親和性タンパク質複合体の精製と青ネイティブPAGEによるそれらの分離のためのプロトコルを提示します。この方法は、異なるタンパク質複合体へのインタラクトームを解決するのに便利です。 

ビデオ: Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled哺乳動物細胞からのタンパク質複合体の親和性のキャプチャ

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

生化学

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

真核細胞からのタンパク質複合体のタンデムアフィニティー精製

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多量-PAGE：生体試料中のキャプチャと解決タンパク質複合体のための方法

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of RNA-protein interactions. These interactions have been traditionally analyzed with polyacrylamide gels generated between two glass plates and samples electrophoresed vertically. However, polyacrylamide gels cast in trays and electrophoresed horizontally offers several advantages. For example, horizontal gels used to analyze complexes between fluorescent RNA substrates and specific proteins can be imaged multiple times as electrophoresis progresses. This provides the unique opportunity to monitor RNA-protein complexes at several points during the experiment. In addition, horizontal gel electrophoresis makes it possible to analyze many samples in parallel. This can greatly facilitate time course experiments as well as analyzing multiple reactions simultaneously to compare different components and conditions. Here we provide a detailed protocol for generating and using horizontal native gel electrophoresis for analyzing RNA-Protein interactions.

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

タンパク質-RNA複合体の検出を向上させるための水平ゲル電気泳動

Native polyacrylamide gel electrophoresis is a fundamental tool of molecular biology that has been used extensively for the biochemical analysis of ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

ネイティブな携帯電話環境におけるコンテキスト固有蛋白質複合体の分割-BioID-プロテオーム解析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 の 1: タンパク質・ タンパク質代謝物の複合体の並列解析のための親和性の浄化をワンステップ

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP+ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b6f (Cyt b6f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

ブルー ネイティブ電気泳動によってチラコイド膜タンパク質複合体の解析

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

タップのタグを使用して葉緑体トランスロコン タンパク質複合体の親和性の浄化

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

蛍光顕微鏡を使用して競争力のある滴定による転写因子の DNA 結合親和性の高感度測定

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

タンパク質の折り畳み、トランスポート、および放射性パルス追跡による生体細胞の劣化の解析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...