MN-S erkennt an Unterstützung von der National Science Foundation, RUI gewähren CHE 1011859. Die Autoren danken der Triad-Massenspektrometrie der Einrichtung an der Universität von North Carolina in Greensboro für die Verwendung des Thermo Fisher Scientific LTQ Orbitrap XL-Massenspektrometer. Die Autoren danken Daniel Todd, Vincent Sica und Brandie Erhmann an der Universität von North Carolina in Greensboro für Anregungen und Kommentare zu dieser Arbeit.

Hinweis: Bitte beachten Sie alle relevanten Sicherheitsdatenblätter (MSDS) vor dem Gebrauch. Quecksilberchlorid ist eine giftige Chemikalie. Persönliche Schutzausrüstung (Handschuhe, Schutzbrille und Labormantel) muss bei der Abgabe ist es und alle damit verbundenen Lösungen getragen werden. Entsorgen Sie die Lösungen in klar beschriftet für Schwermetalle bezeichnet chemische Abfallflaschen. 1. Herstellung von 5 mM Entgastes Ammoniumformiat-Puffer, pH 7,5 <ol><li> Aufzulösen 0,1576 g Ammoniumformiat-Puffer in 450 ml Wasser mit HPLC. Einstellen des pH der obigen Lösung mit 1 M Ameisensäure und 1 M Ammoniumhydroxid auf 7,5. Diese Lösung in einen 500-ml-Messkolben und fügen HPLC-Wasser bis zur Marke, um eine 5 mM Ammoniumformiat-Lösung zu machen. </li><li> Entgasen das 5 mM Ammoniumformiat-Puffer bei einem Vakuum-System für 10 Minuten und Spülung mit Argon. Zweimal wiederholen und Filiallösung unter Argon. Am Tag der Anwendung filtern die Pufferlösung durch einen 0,2 Mikron-Filter vor deme Gebrauch. </li></ol> 2. Herstellung von Quecksilber (II) -chlorid-Lösungen <ol><li> Abwiegen 0,2375 g Quecksilber (II) -chlorid. Löst ihn in 25 ml von 5 mM Ammoniumformiat-Puffer, um eine 0,035 M Quecksilber (II) -chlorid-Lösung zu erzeugen. </li><li> Hinzufügen 0,214 ml von 0,035 M Quecksilber (II) -chlorid-Lösung bis 9.785 ml 5 mM Ammoniumformiat-Puffer, um einen 7,5 x 10 -4 M-Lösung. Decke die 7,5 x 10 -4 M Quecksilber (II) Lösung mit Argongas. </li></ol> 3. Herstellung von CGGC Stammlösung <ol><li> Aufzulösen 2,0 mg des dicysteinyl Tetra CGGC, in 0.118 ml Acetonitril von HPLC-Qualität und fügen 1,0647 ml 5 mM Ammoniumformiat, pH 7,5 Puffer, der in Argon entgast wurde, um eine 5 mM Stammlösung zu ergeben CGGC. </li><li> Fügen Sie 225 ul der 5 mM CGGC Stammlösung zu 1275 ul 5 mM Ammoniumbildung pH 7,5-Puffer, um eine 7,5 x 10 -4 M CGGC Lösung. </li></ol> 4. Vorbereitungverschiedener Reaktions Mischungen von Quecksilber (II) und CGGC <ol><li> Herstellung 1: 0,5-Verhältnis von Quecksilber (II): CGGC Lösung <ol><li> Platzieren 255 ul 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen. Werden 30 &amp; mgr; l von 7,5 x10 -4 M Quecksilber (II) -chlorid-Lösung in 1,5 ml Mikrozentrifugenröhrchen mit dem Ammoniumformiat-Puffer. </li><li> Vortex die Lösung für 10 Sek. Dann fügen Sie 15 ul 7,5 x 10 -4 M CGGC Lösung in den 1,5-ml-Mikro. Vortex die Lösung für 10 Sek. Lassen Sie die Lösung stehen für 10 Minuten vor der Injektion in das Massenspektrometer. </li></ol></li><li> Herstellung von 1: 1-Verhältnis von Quecksilber (II): CGGC Lösung <ol><li> Platzieren 240 ul 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen. Werden 30 &amp; mgr; l von 7,5 x10 -4 M Quecksilber (II) -chlorid-Lösung in 1,5 ml Mikrozentrifugenröhrchen mit dem Ammoniumformiat-Puffer. </li><li> Vortex die Lösung für 10 Sek. DannAdd 30 ul 7,5 x 10 -4 M CGGC Lösung in den 1,5-ml-Mikrozentrifugenröhrchen. Wiederholen Sie in einer ähnlichen Weise, wie in Abschnitt 4.1 beschrieben. </li></ol></li><li> Herstellung von 1: 2-Verhältnis von Quecksilber (II): CGGC Lösung <ol><li> Platzieren 210 ul 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen. Werden 30 &amp; mgr; l von 7,5 x10 -4 M Quecksilber (II) -chlorid-Lösung in 1,5 ml Mikrozentrifugenröhrchen mit dem Ammoniumformiat-Puffer. </li><li> Vortex die Lösung für 10 Sek. Dann fügen Sie 60 ul von 7,5 x 10 -4 M CGGC Lösung in den 1,5-ml-Mikrozentrifugenröhrchen. Wiederholen Sie in einer ähnlichen Weise, wie in Abschnitt 4.1 beschrieben. </li></ol></li></ol> 5. Vorbereitung der MOEL-Stammlösung <ol><li> Man löst 3,5 mg des dicysteinyl Tetrapeptid, MOEL, in 0.145 ml Acetonitril von HPLC-Qualität, um das Peptid zu lösen. Fügen 13,067 ml 5 mM Ammoniumformiat, pH 7,5 Puffer, der in Argon entgast wurde, um 0,5 m zu erzeugenM MOEL-Lösung. </li><li> Vortex die Lösung, bis alle Peptid wird aufgelöst. Hinzufügen von 1,125 ml 0,5 mM MOE-Lösung und 0,375 ml von 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen, eine 7,5 x 10 -5 M MOE Lösung zu ergeben. Vortex, bis gemischt. </li></ol> 6. Herstellung von verschiedenen Reaktions Mischungen von Quecksilber (II) und den MOEL-Lösung <ol><li> Herstellung 1: 0,5-Verhältnis von Quecksilber (II): MOE Lösung <ol><li> Platzieren 255 ul 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen. Werden 30 &amp; mgr; l von 7,5 x10 -4 M Quecksilber (II) -chlorid-Lösung in 1,5 ml Mikrozentrifugenröhrchen mit dem Ammoniumformiat-Puffer. </li><li> Vortex die Lösung für 10 Sek. Dann fügen Sie 15 ul 7,5 x 10 -4 M MOEL-Lösung in die 1,5-ml-Mikrozentrifugenröhrchen. Wiederholen Sie in einer ähnlichen Weise, wie in Abschnitt 4.1 beschrieben. </li></ol></li><li> Herstellung von 1: 1-Verhältnis von Quecksilber (II): MOE Lösung <ol><li> Zeigen 24081; l von 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen. Werden 30 &amp; mgr; l von 7,5 x10 -4 M Quecksilber (II) -chlorid-Lösung in 1,5 ml Mikrozentrifugenröhrchen mit dem Ammoniumformiat-Puffer. </li><li> Vortex die Lösung für 10 Sek. Dann fügen Sie 30 ul 7,5 x 10 -4 M MOEL-Lösung in die 1,5-ml-Mikrozentrifugenröhrchen. Wiederholen Sie in einer ähnlichen Weise, wie in Abschnitt 4.1 beschrieben. </li></ol></li><li> Herstellung von 1: 2-Verhältnis von Quecksilber (II): MOE Lösung <ol><li> Platzieren 210 ul 5 mM Ammoniumformiat, pH 7,5-Puffer in ein 1,5 ml Mikrozentrifugenröhrchen. Werden 30 &amp; mgr; l von 7,5 x10 -4 M Quecksilber (II) -chlorid-Lösung in 1,5 ml Mikrozentrifugenröhrchen mit dem Ammoniumformiat-Puffer. </li><li> Vortex die Lösung für 10 Sek. Dann fügen Sie 60 ul von 7,5 x 10 -4 M MOEL-Lösung in die 1,5-ml-Mikrozentrifugenröhrchen. Wiederholen Sie in einer ähnlichen Weise, wie in Abschnitt 4.1 beschrieben. </li></ol></li></ol> 7. Analyzing Die Reaktionsgemische von Quecksilber (II) und CGGC Proben von Orbitrap ESI-Massenspektrometrie <ol><li> Vorbereiten des ESI-Massenspektrometer 16 <ol><li> Zeichnen Sie 100 ul Eichstandards in einen 500 &amp; mgr; l-Glasspritze. </li><li> Legen Sie die Spritze in der Spritzen Wiege der MS Pumpe, befestigen Sie den Schlauch, und injizieren in die Masse Spektralphotometer. </li><li> Richten Sie den Dateinamen für den Lauf, indem Sie auf das Dateisymbol und geben Sie den Dateinamen. </li><li> Wählen Sie erwerben Daten Schaltfläche in der Datenerfassungsmodul und sammeln Sie 150 Scans. </li><li> Analyse des Chromatogramms, um die Kalibrierungsstandards durch Öffnen Datenverarbeitungsmodul der Software zu überprüfen. Öffnen Sie das Modul, gehen Sie im Menü Datei und wählen Sie &quot;Öffnen&quot;, und wählen Sie die Datei im Dialogfeld. Sicherzustellen, dass die Peaks im Chromatogramm korrelieren mit der Masse, um Verhältnisse der Standards zu berechnen. </li><li> Reinigen Sie den 500 ul Glasspritze durch die Erstellung von 500 ul HPLC-Qualität Methanol, dann verzichten die mEthanol in einem Becherglas. </li><li> Auszuarbeiten 500 ul HPLC-Qualität Methanol in das Glas Spritze und spülen Sie das System gemäß Schritt 7.1.2. </li><li> Wählen Sie die Methode Setup-Modul der Software, um die Parameter einzustellen. Wählen Sie den Scan-Modus-Menü und Identifizierung der Analysator FTMS, und klicken Sie auf &quot;OK&quot;. Dann, indem Sie auf die verschiedenen Symbole auf der Echtzeit-Ansicht Spektrum Seite, die folgenden Parameter: Mantel Gasdurchsatz: 10, Quelle Temperatur: 0, Kapillare Spannung: 37 V, U-Objektiv: 95 V, Spray Spannung: 4,20 kV , Durchflussmenge 10,00 &amp; mgr; l / min, Analyzer: FTMS, Anzahl der Scans: 150. </li></ol></li><li> Laufende CGGC Proben auf ESI-Massenspektrometer <ol><li> Führen Sie das 5 mM Ammoniumformiat pH 7,5 Puffer. <ol><li> Zeigen 500 ul 5 mM Ammoniumformiat-Puffer in die 500 &amp; mgr; l-Glasspritze, legen Sie sie in die Spritze Wiege der MS-Pumpe, und befestigen Sie den Schlauch. </li><li> Pufferlauf durch die Rohrleitung für 1-2 min. </li><li> Richten Sie den Dateinamen für den Laufindem Sie auf das Dateisymbol und geben Sie den Dateinamen. </li><li> Wählen Sie erwerben Schaltfläche Daten in das Modul und sammeln Sie 150 Scans. </li><li> Klicken Sie auf den Schalter zum Sammlung zu stoppen, nachdem 150 Scans gesammelt werden. </li><li> Öffnen Sie den Daten-Browser-Modul, dann gehen Sie im Menü Datei und wählen Sie &quot;Öffnen&quot;, und wählen Sie die Datei im Dialogfeld. Stellen Sie sicher, dass keine Peaks bei 483, 683, 1163 und 1363 m / z vorhanden sind, die das Peptid oder Quecksilber (II) -Peptid-Komplexe ähnlich sind. </li></ol></li><li> Führen Sie das 1: 0,5 Quecksilber (II): CGGC Verhältnis Lösung. <ol><li> Platzieren 250 ul 1: 0,5 Quecksilber (II): CGGC Verhältnis der Probe in die Spritze. </li><li> Legen Sie die Spritze in die Spritzen Wiege der MS Pumpe, befestigen Sie den Schlauch und die Vorrichtung vorzubereiten. </li><li> Wählen Sie einen Dateinamen für die Sicht, indem Sie auf das Dateisymbol und geben Sie den Dateinamen. </li><li> Drücken Sie die acquire Daten Schaltfläche in der Datenerfassungsmodul und sammeln Sie 150 Scans und klicken Sie auf die Schaltfläche Ausführen, um die Sammlung zu stoppen. </li><li> Öffnen Sie das Modul, gehen Sie im Menü Datei und wählen Sie &quot;Öffnen&quot;, und wählen Sie die Datei im Dialogfeld. Stellen Sie sicher, dass das Chromatogramm enthält Peaks, einschließlich der für den CGGC Peptid allein. </li><li> Durch Absaugen mit 500 ul Ammoniumformiat-Puffer, und dann Abgeben der Ammoniumformiat-Puffer in ein Becherglas zu waschen die Spritze. </li><li> Wählen Sie das Abfall Taste auf der MS und spülen Sie den Schlauch dreimal mit 500 &amp; mgr; l Ammoniumformiat-Puffer. </li><li> Durch Absaugen mit 500 ul Methanol und dann Abgeben der Methanol in ein Becherglas zu waschen die Spritze. </li><li> Spülen Sie die Schläuche einmal mit 500 ul Methanol. </li><li> Wählen Lastdetektor Taste auf die MS. </li><li> Gib 500 ul der Ammoniumformiat-Puffer, um die Spritze. </li><li> Legen Sie die Spritze in die Spritzen Wiege der MS Pumpe, befestigen Sie den Schlauch und die Vorrichtung vorzubereiten. </li><li> Wählen Sie einen Dateinamen für den Puffer, indem Sie auf das Dateisymbol und geben Sie den Dateinamen ausführen. </li><li> Drücken Siedie acquire Datentaste und sammeln Sie 150 Scans und klicken Sie auf die Stopp-Taste RUN. </li><li> Öffnen Sie den Daten-Browser-Modul, gehen Sie im Menü Datei und wählen Sie &quot;Öffnen&quot;, und wählen Sie die Datei im Dialogfeld. Stellen Sie sicher, dass das Chromatogramm ist nichtig von Gipfeln aus dem vorherigen Hg: CGGC Lauf. </li></ol></li><li> Führen Sie den 1: 1-Quecksilber (II): CGGC Verhältnis Lösung. <ol><li> Platzieren 250 ul 1: 1 Quecksilber (II): CGGC Verhältnis Probe in die Spritze. </li><li> Wiederholt in einer ähnlichen Weise wie für die Schritte 7.2.2.2 bis 7.2.2.15 beschrieben. </li></ol></li><li> Führen Sie den 1: 2 Quecksilber (II): CGGC Verhältnis Lösung <ol><li> Platzieren 250 ul 1: 2 Quecksilber (II): CGGC Konzentration Probe in die Spritze. </li><li> Wiederholt in einer ähnlichen Weise wie für die Schritte 7.2.2.2 bis 7.2.2.15 beschrieben. </li></ol></li></ol></li></ol> 8. Die Analyse der Reaktionsgemische von Merkur und MOEL Proben von Orbitrap ESI-Massenspektrometrie <ol><li> Laufende MOEL Proben auf ESI-Massenspektrometer <ol><li> Wiederholen Sie die Analyseverfahren (Schritte von 7,1 bis 7,2) mit MOEL Proben und Reaktionsgemische von Quecksilber (II) und den MOEL an verschiedenen stöchiometrischen Verhältnissen. </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.

Das hydrophobe dicysteinyl Tetra CGGC (C 10 H 18 N 4 O 5 S 2; Mw = 338) (Figur 1) bildet Komplexe mit Quecksilber (II), wie in Abbildung 2 dargestellt und in Tabelle 1 Zusätzlich Peptid-Dimere und Trimere bildet. inkrementell als die Menge des Peptids erhöht sich in der Reaktionsmischung. Wie durch die m / z-Werte der zugehörigen Dimere [(2M + H) + = 677] und Trimere gezeigt [(3M + <su...

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> Eine Studie wurde durchgeführt, um die mögliche Quecksilber-Peptid-Komplex-Zusammensetzung für zwei Tetrapeptiden, CGGC und den MOEL (Abbildung 1) durch ESI-Massenspektrometrie zu charakterisieren. Komplexe von Quecksilber (II) mit CGGC oder MOE wurden durch Umsetzung der Mischungen aus Quecksilber untersucht (II) und Peptidlösungen in drei verschiedenen Molverhältnissen: 1: 0,5, 1: 1 und 1: 2 (Quecksilber (II): Peptid) . Die Konzentration von Quecksilber (II)...

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.

Eine Studie der Komplexierung von Quecksilber (II) mit Dicysteinyl Tetrapeptide durch Elektrospray-Ionisations-Massenspektrometrie

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 de Cartagena UDEC

Research

JoVE Journal

Chemistry

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

7.8K Aufrufe.  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.

Serie: 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.

Bestimmung der Gas-Phase Aciditäten von Oligopeptides

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

Forschung

Chemie

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.

Produktion von Disulfid-stabilisierte Transmembrane Peptid-Komplexe für Structural Studies

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.

Compact Quantum Dots für Einzelmolekül-Imaging

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

Ingenieurwesen

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.

Zeitaufgelöste Elektrospray-Ionisations Wasserstoff-Deuterium-Austausch-Massenspektrometrie zur Struktur Protein Studium und Dynamik

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

Biochemie

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.

Massenspektrometrische Ansätze zur Proteinstruktur und Wechselwirkungen zu untersuchen in lyophilisierte Pulver

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.

Eine Wasserstoff-Deuterium-Austausch-Massenspektrometrie -Plattform (HDX-MS) zur Untersuchung biosynthetischer Peptidenzyme

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.

Ionen-Mobilitäts-Massenspektrometrie-Techniken zur Bestimmung der Struktur und Mechanismen der Metallionenerkennung und Redox-Aktivität von Metallbindungsoligopeptiden

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.

Analyse großer Proteinkomplexe von Structural-Massenspektrometrie

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Biologie

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.

Thermochemische Untersuchungen von ni(II) und Zn(II) ternären Komplexen mittels Ionenmobilitäts-Massenspektrometrie

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.

Profiling von Methyltransferasen und andere S-Adenosyl- L-Homocystein-bindende Proteine ​​von 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 ...

Eine Studie der Komplexierung von Quecksilber (II) mit Dicysteinyl Tetrapeptide durch Elektrospray-Ionisations-Massenspektrometrie

Zusammenfassung

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Thermochemische Untersuchungen von ni(II) und Zn(II) ternären Komplexen mittels Ionenmobilitäts-Massenspektrometrie

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Eine Studie der Komplexierung von Quecksilber (II) mit Dicysteinyl Tetrapeptide durch Elektrospray-Ionisations-Massenspektrometrie

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Profiling von Methyltransferasen und andere S-Adenosyl-_L-Homocystein-bindende Proteine von Capture Compound Mass Spectrometry (CCMS)