Name: a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry
Uploaded: 2016-01-08T13:00:01.000+00:00
Duration: 12 min 59 s
Description: Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

MN-Sは全米科学財団からの支援を承認、RUIは、著者は感謝してサーモフィッシャーサイエンティフィックLTQオービトラップXL質量分析計を使用するためにノースカロライナ大学グリーンズボロ校でトライアド質量分析施設を承認CHE 1011859.を付与します。作者はこの作品に関する有用な提案やコメントのためにノースカロライナ大学グリーンズボロ校でダニエル・トッド、ヴィンセントシーカ、およびBrandie Erhmannに感謝します。

注：使用する前に、関連するすべての物質安全データシート（MSDS）を参照してください。塩化水銀は有毒化学物質です。それと関連するすべてのソリューションを渡す際に個人用保護具（手袋、安全ゴーグル、および白衣）を着用しなければなりません。重金属のために指定された明確に表示化学廃液ボトル内のソリューションを処分。 5 mMの脱気したギ酸アンモニウム緩衝液（pH7.5）の調製<ol><li> HPLCグレードの水450ミリリットルにギ酸アンモニウム緩衝液の0.1576グラムを溶解します。 1 Mギ酸および7.5から1 M水酸化アンモニウムで上記溶液のpHを調整します。 500ミリリットルのメスフラスコに、このソリューションを移し、5 mMのギ酸アンモニウム溶液を作製するために検量線にHPLC水を追加します。 </li><li>アルゴンで10分間パージするための真空システムの下で5 mMのギ酸アンモニウム緩衝液を脱気。アルゴン下で二回繰り返して、ストア・ソリューション。使用の日に、血友病0.2ミクロンフィルターを通して緩衝液をフィルタE使用。 </li></ol>水銀（II）塩化物溶液の調製<ol><li> 0.2375グラムの水銀（II）塩化物を秤量。 0.035 M水銀（II）塩化物溶液を生成するために5mMのギ酸アンモニウム緩衝液25ml中に溶解します。 </li><li> 7.5×10 -4 Mのソリューションを作成するために、9.785ミリリットルの5mMのギ酸アンモニウムバッファーに0.214ミリリットル0.035 M水銀（II）塩化物溶液を追加します。アルゴンガスを7.5×10 -4 Mの水銀（II）溶液をブランケット。 </li></ol> CGGCストック溶液の調製<ol><li> HPLCグレードのアセトニトリルの0.118ミリリットルで、dicysteinylテトラペプチド、CGGC 2.0mgのを溶解した後、5 mMのギ酸アンモニウム、5mMのCGGC原液を得アルゴンで脱気されたpHが7.5の緩衝液1.0647ミリリットルを追加します。 </li><li> 7.5×10 -4 M CGGC溶液を得5mMのアンモニウム形成のpHの1,275μlの7.5バッファに5 mMのCGGC原液の225μLを加えます。 </li></ol> 4.準備水銀（II）とCGGCの種々の反応混合物<ol><li>水銀の0.5比（II）：CGGC液1の調製<ol><li> 1.5mlの微小管に5 mMのギ酸アンモニウム、pH7.5の緩衝液を255μLを入れます。ギ酸アンモニウム緩衝液1.5 mlマイクロチューブに7.5×10 -4 Mの水銀（II）塩化物溶液の30μlのを追加します。 </li><li> 10秒のためのソリューションをVORTEX。その後、1.5mlの微小に7.5×10 -4 M CGGC溶液を15μlを添加します。 10秒のためのソリューションをVORTEX。ソリューションは、質量分析計に注入前に10分間放置してみましょう。 </li></ol></li><li>水銀の1の比率（II）：CGGC液1の調製<ol><li> 1.5mlの微小管に5 mMのギ酸アンモニウム、pH7.5の緩衝液を240μLを入れます。ギ酸アンモニウム緩衝液1.5 mlマイクロチューブに7.5×10 -4 Mの水銀（II）塩化物溶液の30μlのを追加します。 </li><li> 10秒のためのソリューションをVORTEX。そして、1.5ミリリットルマイクロ遠心チューブに7.5×10 -4 M CGGC溶液を30μlを添加します。 4.1節で説明したと同様に繰り返します。 </li></ol></li><li>水銀の2比（II）：CGGC液1の調製<ol><li> 1.5mlの微小管に5 mMのギ酸アンモニウム、pH7.5の緩衝液を210μLを入れます。ギ酸アンモニウム緩衝液1.5 mlマイクロチューブに7.5×10 -4 Mの水銀（II）塩化物溶液の30μlのを追加します。 </li><li> 10秒のためのソリューションをVORTEX。その後、1.5mlの微小管に7.5×10 -4 M CGGC溶液を60μlを添加します。 4.1節で説明したと同様に繰り返します。 </li></ol></li></ol> CEEC原液の5準備<ol><li>ペプチドを溶解させ、HPLCグレードのアセトニトリルの0.145ミリリットルで、dicysteinylテトラペプチド、CEECの3.5 mgの溶解。その後5mMのギ酸アンモニウム、0.5メートルを生成するためにアルゴンで脱ガスされたpHが7.5の緩衝液13.067ミリリットルを加えますM CEECソリューション。 </li><li>すべてのペプチドが溶解するまで溶液をボルテックス。 7.5×10 -5 M CEEC溶液を得、1.5 mlのマイクロ遠心チューブに、1.125ミリリットル0.5 mMのをpH7.5のバッファーをCEEC液および5mMのギ酸アンモニウムの0.375ミリリットルを追加します。ボルテックス混合されるまで。 </li></ol>水銀（II）とCEECソリューションの様々な反応混合物の調製6。 <ol><li> 1の調製：水銀（II）の比は0.5：CEEC溶液<ol><li> 1.5mlの微小管に5 mMのギ酸アンモニウム、pH7.5の緩衝液を255μLを入れます。ギ酸アンモニウム緩衝液1.5 mlマイクロチューブに7.5×10 -4 Mの水銀（II）塩化物溶液の30μlのを追加します。 </li><li> 10秒のためのソリューションをVORTEX。その後、1.5mlの微小管に7.5×10 -4 M CEEC溶液を15μlを添加します。 4.1節で説明したと同様に繰り返します。 </li></ol></li><li>水銀（II）の1：1の比：1の調製CEECソリューション<ol><li> 240を配置81; 1.5ミリリットルマイクロ遠心チューブに5 mMのギ酸アンモニウム、pH7.5の緩衝液のリットル。ギ酸アンモニウム緩衝液1.5 mlマイクロチューブに7.5×10 -4 Mの水銀（II）塩化物溶液の30μlのを追加します。 </li><li> 10秒のためのソリューションをVORTEX。その後、1.5mlの微小管に7.5×10 -4 M CEEC溶液を30μlを添加します。 4.1節で説明したと同様に繰り返します。 </li></ol></li><li>水銀（II）の2比：1の調製CEECソリューション<ol><li> 1.5mlの微小管に5 mMのギ酸アンモニウム、pH7.5の緩衝液を210μLを入れます。ギ酸アンモニウム緩衝液1.5 mlマイクロチューブに7.5×10 -4 Mの水銀（II）塩化物溶液の30μlのを追加します。 </li><li> 10秒のためのソリューションをVORTEX。その後、1.5mlの微小管に7.5×10 -4 M CEEC溶液を60μlを添加します。 4.1節で説明したと同様に繰り返します。 </li></ol></li></ol> 7. ANALYオービトラップESI質量分析による水銀（II）とCGGCサンプルの反応混合物をシュッ<ol><li> ESI質量分析計16の準備 <ol><li> 500μlのガラスシリンジに校正標準の100μLを描画します。 </li><li> 、MSポンプのシリンジクレードルに注射器を置き、チューブを取り付け、質量分光光度計の中に注入します。 </li><li>ファイルのアイコンを選択し、ファイル名を入力して実行するためのファイル名を設定します。 </li><li>データ取得モジュールで取得するデータボタンを選択し、150スキャンを収集します。 </li><li>ソフトウェアのデータ処理モジュールを開くことによって、較正基準を検証するクロマトグラムを分析します。モジュールを開き、メニューをファイルに移動して、「オープン」を選択し、ダイアログボックスでファイルを選択します。クロマトグラムのピークが基準の比率を質量電荷に相関していることを確認します。 </li><li> 500μlのHPLCグレードのメタノールを描画することにより500μlのガラスシリンジをきれいにして、Mを分配ビーカーにエタノール。 </li><li>ガラスシリンジにHPLCグレードのメタノール500μlのを描画し、ステップ7.1.2のとおりにシステムをフラッシュします。 </li><li>パラメータを設定するためのソフトウェアの方法のセットアップ・モジュールを選択します。スキャンモードメニューを選択し、FTMSなどの分析器を特定し、「OK」をクリックしてください。シースガス流量：10、ソース温度：0、キャピラリー電圧：37 V、チューブレンズ：95 V、スプレー電圧：4.20 kVのその後のリアルタイムビュースペクトルページのさまざまなアイコンをクリックすることで、以下のパラメータを設定します、フローは10.00μL/ minで、アナライザを評価：FTMS、スキャン数：150。 </li></ol></li><li> ESI質量分析計でCGGCサンプルを実行します<ol><li> 5 mMのギ酸アンモニウムpH7.5のバッファーを実行します。 <ol><li> 、500μlのガラスシリンジに5 mMのギ酸アンモニウム緩衝液500μlを置き、MSポンプのシリンジクレードルにそれを置き、チューブを取り付けます。 </li><li> 1-2分間チューブを介してバッファを実行します。 </li><li>ランのファイル名を設定しますファイルのアイコンを選択し、ファイル名を入力して。 </li><li>モジュール内の取得データ]ボタンを選択し、150スキャンを収集します。 </li><li> 150スキャンを収集した後に収集を停止するには[実行]ボタンをクリックします。 </li><li>データブラウザモジュールを開き、メニューをファイルに移動して、「オープン」を選択し、ダイアログボックスでファイルを選択します。ペプチドまたは水銀（II） - ペプチド複合体に似ていることに何の483、683、1163および1363のピークのm / zが存在しないことを確認します。 </li></ol></li><li> 0.5水銀（II）：CGGC比液1を実行します。 <ol><li> 0.5水銀（II）：注射器への試料のCGGC比1の250μLを入れます。 </li><li>チューブ、および素数装置を取り付け、MSポンプのシリンジクレードルに注射器を置きます。 </li><li>ファイルのアイコンを選択し、ファイル名を入力して実行するためのファイル名を選択します。 </li><li>データ取得モジュールで取得するデータボタンを押して、150スキャンを収集し、収集を停止するには[実行]ボタンをクリックします。 </li><li>モジュールを開き、メニューをファイルに移動して、「オープン」を選択し、ダイアログボックスでファイルを選択します。クロマトグラムは、単独でCGGCペプチドのための1つを含むピークが含まれていることを確認します。 </li><li> 500μlのギ酸アンモニウム緩衝液で吸引した後、ビーカーにギ酸アンモニウムバッファーを分配することによって、注射器を洗浄します。 </li><li> MSの廃棄物のボタンを選択し、チューブに500μlのギ酸アンモニウム緩衝液で3回洗浄します。 </li><li> 500μlのメタノールで吸引した後、ビーカーにメタノールを分配することによって、注射器を洗浄します。 </li><li> 500μlのメタノールでチューブ1時間をフラッシュします。 </li><li> MSの負荷検出器のボタンを選択します。 </li><li>シリンジにギ酸アンモニウム緩衝液500μlを追加します。 </li><li>チューブ、および素数装置を取り付け、MSポンプのシリンジクレードルに注射器を置きます。 </li><li>ファイルのアイコンを選択し、ファイル名を入力して実行し、バッファのファイル名を選択します。 </li><li>押す取得データボタンと150スキャンを収集した後、停止実行ボタンをクリックします。 </li><li>データブラウザモジュールを開き、メニューをファイルに移動して、「オープン」を選択し、ダイアログボックスでファイルを選択します。 CGGCラン：クロマトグラムは、前水銀からのピークの空隙であることを確認します。 </li></ol></li><li> 1水銀（II）：CGGC比液1を実行します。 <ol><li>シリンジにCGGC比サンプル：1の水銀（II）：1の250μLを入れます。 </li><li> 7.2.2.15までの流れ7.2.2.2で説明したように、同様に繰り返します。 </li></ol></li><li> 2水銀（II）：CGGC比液1を実行します。 <ol><li>シリンジにCGGC濃度サンプル：2水銀（II）：1の250μLを入れます。 </li><li> 7.2.2.15までの流れ7.2.2.2で説明したように、同様に繰り返します。 </li></ol></li></ol></li></ol> 8.オービトラップESI質量分析法による水銀およびCEECサンプルの反応混合物の分析<ol><li> ESI質量分析計でCEECサンプルを実行します<ol><li>繰り返し分析手順（ステップ7.1〜7.2）CEECサンプルや様々な化学量論比での水銀（II）とCEECの反応混合物を使用。 </li></ol></li></ol>

<ol>
	<li>Clifton, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+exposure+and+public+health.">Mercury exposure and public health.</a> Pediatr. Clin. N. Am. 54, 237-269 (2007).</li><li>Andersen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+Recent+Developments+in+Chelation+Treatment+of+Metal+Intoxication.">Principles and Recent Developments in Chelation Treatment of Metal Intoxication.</a> Chem. Rev. 99, 2683-2710 (1999).</li><li>Aposhian, H. V., Maiorino, R. M., Gonzalez-Ramirez, D., Zuniga-Charles, M., Xu, Z., Hurlbut, J. M., Junco-Munoz, P., Dart, R. C., Aposhian, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+heavy+metals+by+newer,+therapeutically+useful+chelating+agents.">Mobilization of heavy metals by newer, therapeutically useful chelating agents.</a> Toxicology. 97, 23-38 (1995).</li><li>Flora, S. J. S., Pachauri, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chelation+in+Metal+Intoxication.">Chelation in Metal Intoxication.</a> Int. J. Environ. Res. Public Health. 7, 2745-2788 (2010).</li><li>Campbell, J. R., Clarkson, T. W., Omar, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+use+of+2,3-dimercaptopropane-1-sulfonate+in+two+cases+of+inorganic+mercury+poisoning.">The therapeutic use of 2,3-dimercaptopropane-1-sulfonate in two cases of inorganic mercury poisoning.</a> JAMA. 256, 3127-3130 (1986).</li><li>Rooney, J. P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+thiols,+dithiols,+nutritional+factors+and+interacting+ligands+in+the+toxicology+of+mercury.">The role of thiols, dithiols, nutritional factors and interacting ligands in the toxicology of mercury.</a> Toxicology. 234, 145-156 (2007).</li><li>Lin, X., Brooks, J., Bronson, M., Ngu-Schwemlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evalution+of+the+association+of+mercury+(II)+with+some+dicysteinyl+tripeptides.">Evalution of the association of mercury (II) with some dicysteinyl tripeptides.</a> Bioorg. Chem. 44, 8-18 (2012).</li><li>Ngu-Schwemlein, M., Lin, X., Rudd, B., Bronson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+ESI+mass+spectrometric+analysis+of+the+association+of+mercury(II)+with+multi-cysteinyl+peptides.">Synthesis and ESI mass spectrometric analysis of the association of mercury(II) with multi-cysteinyl peptides.</a> J. Inorg. Biochem. 133, 8-23 (2014).</li><li>Winther, J. R., Thorpe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+thiols+and+disulfides.">Quantification of thiols and disulfides.</a> Biochimica et. Biophysica Acta. 1840, 838-846 (2014).</li><li>D'Agstino, A., Colton, R., Traeger, J. C., Cantry, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Electrospray+Mass+Spectrometric+Study+of+Organomercury+(II)+and+Mercuric+Interactions+with+Peptides+Involving+Cysteinyl+Ligands.">An Electrospray Mass Spectrometric Study of Organomercury (II) and Mercuric Interactions with Peptides Involving Cysteinyl Ligands.</a> Eur. Mass Spectrom. , 273-285 (1990).</li><li>Hofstadler, S. A., Sannes-Lowery, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+ESI-MS+in+drug+discovery:+interrogation+of+noncovalent+complexes.">Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes.</a> Nature Reviews Drug Discovery. 5, 585-595 (2006).</li><li>Rubino, F. M., Verduci, C., Giampiccolo, R., Pulvirenti, S., Brambilla, G., Columbi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Characterization+of+Homo-+and+Heterodimeric+Mercury+(II)-bis-thiolates+of+Some+Biologically+Relevant+Thiols+by+Electrospray+Ionization+and+Triple+Quadruple+Tandem+Mass+Spectrometry.">Molecular Characterization of Homo- and Heterodimeric Mercury (II)-bis-thiolates of Some Biologically Relevant Thiols by Electrospray Ionization and Triple Quadruple Tandem Mass Spectrometry.</a> J. Am. Soc. Mass Spectrom. 15, 288-300 (2003).</li><li>Krupp, E. M., Milne, B. F., Mestrot, A., Meharg, A. A., Feldmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+into+mercury+bound+to+biothiols:+structural+identification+using+ESI-ion-trap+MS+and+introduction+of+a+method+for+their+HPLC+separation+with+simultaneous+detection+by+ICP-MS+and+and+ESI-MS.">Investigation into mercury bound to biothiols: structural identification using ESI-ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and and ESI-MS.</a> Anal. Bioanal. Chem. 390, 1753-1764 (2008).</li><li>Schauml&#246;ffel, D., Tholey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+directions+of+electrospray+mass+spectrometry+for+elemental+speciation+analysis.">Recent directions of electrospray mass spectrometry for elemental speciation analysis.</a> Anal. Bioanal. Chem. 400, 1645-1652 (2011).</li><li>Patiny, L., Borel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChemCalc:+a+building+block+for+tomorrow's+chemical+infrastructure.">ChemCalc: a building block for tomorrow's chemical infrastructure.</a> J. Chem. Inf. Model. 53, 1223-1228 (2013).</li><li>Thermo Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xcaibur Versions 2.1.0-2.3.0 Data Acquisition and Processing User Guide. Revision E. United States. , (2012).</li><li>Falcone, G., Foti, C., Gianguzza, A., Giuffr&#232;, O., Napoli, A., Pettignano, A., Piazzese, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequestering+ability+of+some+chelating+agents+towards+methylmercury(II).">Sequestering ability of some chelating agents towards methylmercury(II).</a> Anal. Bioanal. Chem. 405 (2), 881-893 (2013).</li><li>Mah, V., Jalilehvand, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+Complex+Formation+with+Mercury(II)+in+Aqueous+Solution+at+Physiological+pH.">Glutathione Complex Formation with Mercury(II) in Aqueous Solution at Physiological pH.</a> Chem. Res. Toxicol. 23, 1815-1823 (2010).</li></ol>

There is no disclosure for the work reported.

疎水dicysteinylテトラペプチドCGGC（C 10 H 18 N 4 O 5 S 2; = 338 MW）は、図2 及び表1に示すように（ 図1）は 、水銀（II）と複合体を形成しさらに、ペプチド二量体および三量体を形成します。増分反応混合物中のペプチドの量が増加します。 [+ = 1015（3M + H）]のm / z関連する二量体の値[（2...

Current clinical drugs prescribed for chelation therapy of mercury poisoning1 contain thiol group(s), which is/are responsible for binding and sequestering mercury ions2,3. However, studies have shown that these small thiol compounds [dimercaptosuccinic acid (DMSA) and dimercaptopropane-sulfonic acid (DMPS)] are not optimal for mercury chelation therapy4-6. Therefore, there is a need to understand the association and complex formation tendencies of mercury with thiols to enhance the rational drug design of thiol compounds for mercury chelation. Recently, we reported that n-alkyl and aryl dicysteinyl tripeptides with dithiol groups can serve as effective "double anchors" to accommodate the coordination sites of mercury(II) to form 1:1 mercury(II):peptide and 1:2 mercury(II):(peptide)2 complexes7. Additionally, we studied the effect of increasing cysteinyl residues on complex type formations8. In this study, we investigate the association of mercury(II) with two dicysteinyl tetrapeptides, where the cysteinyl residues are separated by two amino acid residues. In order to evaluate the effect of auxiliary binding groups for mercury, the intervening amino acids are either two glycine (unsubstituted) residues or two glutamic acid (gamma-carboxylated) residues. 
The reaction of cysteinyl peptide with mercury(II) requires conditions that will prevent the oxidation of the cysteinyl thiol groups to form disulfide bonds9. Moreover, the association of mercury(II) with cysteinyl peptides to form various types of mercury-peptide complexes is dependent on the initial ratio of mercury(II): peptide in the reaction mixture7,8. The types of mercury-peptide complexes formed in these reaction mixtures can be analyzed by soft-ionization mass spectroscopy, which is a sensitive analytical tool for determining species interactions between metal ions and peptides10-14. Accordingly, it will provide a profile of the various types of mercuriated peptide adducts that are formed under a specified electrospray ionization condition. Here, we will show how cysteinyl peptides and mercury(II) chloride solutions can be prepared in degassed ammonium formate buffer solution blanketed with argon to minimize oxidation. By reacting varying mole equivalents of mercury(II) with dicysteinyl tetrapeptides, we will show how the initial ratio of mercury(II):peptide has an effect on the types of complexes formed. We will also show how electrospray ionization (ESI) mass spectrometry can be used as a characterization tool to provide an accurate stoichiometry of mercury to peptide in the complexes formed. The associated video protocol will demonstrate the experimental conditions for preparing the mercury complexes, the procedure for analyzing the reaction mixtures under specified electrospray ionization conditions, and the characterization of the stoichiometries of mercury(II)-dicysteinyl tetrapeptide complexes, based on the distinct mercury isotope distribution patterns, by using the ChemCal program15. It is intended to assist those who are interested in using ESI orbitrap mass spectrometry to analyze various complexes formed by metal ions that exist in different isotopic forms.

<table><tbody><tr><td>Mercury(II) chloride</td><td>Sigma-Aldrich</td><td>429724</td><td>Highly toxic</td></tr><tr><td>Ammonium formate</td><td>Sigma-Aldrich</td><td>516961</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher</td><td>A512-P500</td><td></td></tr><tr><td>HPLC water</td><td>Fisher</td><td>W5-4</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Fisher</td><td>BP2405-1</td><td></td></tr><tr><td>HPLC Methanol</td><td>Fisher</td><td>A452-4</td><td></td></tr></tbody></table>

a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

<html>研究は、ESI質量分析により2テ ​​トラペプチド、CGGC及びCEEC（ 図1）のための可能な水銀-ペプチド複合体組成物を特徴づけるために実施しました。 1：0.5、1：1、及び1：2（水銀（II）：ペプチド）CGGCまたはCEECと水銀の複合体（II）は、三つの異なるモル比での水銀の混合物（II）とペプチド溶液を反応させて調べました。水銀の濃度（II）が7.5×10 <sup...

Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

エレクトロスプレーイオン化質量分析法によるDicysteinylテトラペプチドと水銀（II）の錯体の研究

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

Universidad San Sebastian

Research

JoVE Journal

Chemistry

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

7.8K ビュー.  University of North Carolina at Greensboro. The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

ビデオ: A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

オリゴペプチドの気相酸性度の決定

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

化学

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Compact Quantum Dots for Single-molecule Imaging

Single-molecule imaging is an important tool for understanding the mechanisms of biomolecular function and for visualizing the spatial and temporal heterogeneity of molecular behaviors that underlie cellular biology 1-4. To image an individual molecule of interest, it is typically conjugated to a fluorescent tag (dye, protein, bead, or quantum dot) and observed with epifluorescence or total internal reflection fluorescence (TIRF) microscopy. While dyes and fluorescent proteins have been the mainstay of fluorescence imaging for decades, their fluorescence is unstable under high photon fluxes necessary to observe individual molecules, yielding only a few seconds of observation before complete loss of signal. Latex beads and dye-labeled beads provide improved signal stability but at the expense of drastically larger hydrodynamic size, which can deleteriously alter the diffusion and behavior of the molecule under study.
Quantum dots (QDs) offer a balance between these two problematic regimes. These nanoparticles are composed of semiconductor materials and can be engineered with a hydrodynamically compact size with exceptional resistance to photodegradation 5. Thus in recent years QDs have been instrumental in enabling long-term observation of complex macromolecular behavior on the single molecule level. However these particles have still been found to exhibit impaired diffusion in crowded molecular environments such as the cellular cytoplasm and the neuronal synaptic cleft, where their sizes are still too large 4,6,7.
Recently we have engineered the cores and surface coatings of QDs for minimized hydrodynamic size, while balancing offsets to colloidal stability, photostability, brightness, and nonspecific binding that have hindered the utility of compact QDs in the past 8,9. The goal of this article is to demonstrate the synthesis, modification, and characterization of these optimized nanocrystals, composed of an alloyed HgxCd1-xSe core coated with an insulating CdyZn1-yS shell, further coated with a multidentate polymer ligand modified with short polyethylene glycol (PEG) chains (Figure 1). Compared with conventional CdSe nanocrystals, HgxCd1-xSe alloys offer greater quantum yields of fluorescence, fluorescence at red and near-infrared wavelengths for enhanced signal-to-noise in cells, and excitation at non-cytotoxic visible wavelengths. Multidentate polymer coatings bind to the nanocrystal surface in a closed and flat conformation to minimize hydrodynamic size, and PEG neutralizes the surface charge to minimize nonspecific binding to cells and biomolecules. The end result is a brightly fluorescent nanocrystal with emission between 550-800 nm and a total hydrodynamic size near 12 nm. This is in the same size range as many soluble globular proteins in cells, and substantially smaller than conventional PEGylated QDs (25-35 nm).

We describe the preparation of colloidal quantum dots with minimized hydrodynamic size for single-molecule fluorescence imaging. Compared to conventional quantum dots, these nanoparticles are similar in size to globular proteins and are optimized for single-molecule brightness, stability against photodegradation, and resistance to nonspecific binding to proteins and cells.

1分子蛍光イメージングのためのコンパクトな量子ドット

Single-molecule imaging is an important tool for  understanding the mechanisms of biomolecular function and for visualizing the  spatial and temporal ...

工学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

生化学

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

質量分析は、凍結乾燥粉末中のタンパク質の構造と相互作用を研究するためのアプローチ

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and enzyme-substrate interactions. Among its many benefits, HDX-MS consumes only small amounts of material, can be performed under near native conditions without the need for enzyme/substrate labeling, and can provide spatially resolved information on enzyme conformational dynamics&#8722;even for large enzymes and multiprotein complexes. The method is initiated by the dilution of the enzyme of interest into buffer prepared in D2O. This triggers the exchange of protium in peptide bond amides (N-H) with deuterium (N-D). At the desired exchange time points, reaction aliquots are quenched, the enzyme is proteolyzed into peptides, the peptides are separated by ultra-performance liquid chromatography (UPLC), and the change in mass of each peptide (due to the exchange of hydrogen for deuterium) is recorded by MS. The amount of deuterium uptake by each peptide is strongly dependent on the local hydrogen bonding environment of that peptide. Peptides present in very dynamic regions of the enzyme exchange deuterium very rapidly, whereas peptides derived from well-ordered regions undergo exchange much more slowly. In this manner, the HDX rate reports on local enzyme conformational dynamics. Perturbations to deuterium uptake levels in the presence of different ligands can then be used to map ligand binding sites, identify allosteric networks, and to understand the role of conformational dynamics in enzyme function. Here, we illustrate how we have used HDX-MS to better understand the biosynthesis of a type of peptide natural products called lanthipeptides. Lanthipeptides are genetically encoded peptides that are post-translationally modified by large, multifunctional, conformationally dynamic enzymes that are difficult to study with traditional structural biology approaches. HDX-MS provides an ideal and adaptable platform for investigating the mechanistic properties of these types of enzymes.

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide synthetases catalyze multistep reactions during the biosynthesis of peptide natural products. Here, we describe a continuous, bottom-up, hydrogen-deuterium exchange mass spectrometry (HDX-MS) workflow that can be employed to study the conformational dynamics of lanthipeptide synthetases, as well as other similar enzymes involved in peptide natural product biosynthesis.

ペプチド生合成酵素を調べた水素重水素交換質量分析(HDX-MS)プラットフォーム

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

金属イオン認識の構造とメカニズムを決定するイオン移動質量分析技術と金属結合オリゴペプチドの酸化還元活性

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

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 ...

生物学

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

イオン移動度質量分析法を用いたNi(II)およびZn(II)三元錯体の熱化学的研究

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル- L</subキャプチャの複合質量分析法（CCMS）別> -ホモシステイン結合タンパク質

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

エレクトロスプレーイオン化質量分析法によるDicysteinylテトラペプチドと水銀（II）の錯体の研究

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この動画の章

オリゴペプチドの気相酸性度の決定

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

1分子蛍光イメージングのためのコンパクトな量子ドット

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

質量分析は、凍結乾燥粉末中のタンパク質の構造と相互作用を研究するためのアプローチ

ペプチド生合成酵素を調べた水素重水素交換質量分析(HDX-MS)プラットフォーム

金属イオン認識の構造とメカニズムを決定するイオン移動質量分析技術と金属結合オリゴペプチドの酸化還元活性

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

イオン移動度質量分析法を用いたNi(II)およびZn(II)三元錯体の熱化学的研究

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル-_{L -ホモシステイン結合タンパク質}

エレクトロスプレーイオン化質量分析法によるDicysteinylテトラペプチドと水銀（II）の錯体の研究

要約

さらに動画を探す

この動画の章

オリゴペプチドの気相酸性度の決定

構造研究のためのジスルフィド安定化膜貫通ペプチド複合体の生産

1分子蛍光イメージングのためのコンパクトな量子ドット

時間分解エレクトロスプレーイオン化水素 - 重水素交換質量分析タンパク質の構造とダイナミクスを学ぶために

質量分析は、凍結乾燥粉末中のタンパク質の構造と相互作用を研究するためのアプローチ

ペプチド生合成酵素を調べた水素重水素交換質量分析(HDX-MS)プラットフォーム

金属イオン認識の構造とメカニズムを決定するイオン移動質量分析技術と金属結合オリゴペプチドの酸化還元活性

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

イオン移動度質量分析法を用いたNi(II)およびZn(II)三元錯体の熱化学的研究

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル- L -ホモシステイン結合タンパク質

メチルトランスフェラーゼのプロファイリングおよびその他の S -アデノシル-_{L -ホモシステイン結合タンパク質}