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>

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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.

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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mercury(II) chloride</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">429724</td>
			<td style="width:167px;">Highly toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium formate</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">516961</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formic acid</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F0507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium hydroxide</td>
			<td style="width:71px;">Fisher</td>
			<td>A512-P500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC water</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">W5-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC Acetonitrile</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2405-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC Methanol</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">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

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

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

Visualisierung von Ambient-Massenspektrometrie mit der Verwendung von Schlieren Fotografie

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Forschung

Chemie

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Fizzy Extraktion von flüchtigen organischen Verbindungen kombiniert mit atmosphärischem Druck Chemische Ionisation Quadrupol Massenspektrometrie

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Echtzeit-Atem-Analyse mithilfe von sekundären Nanoelectrospray Ionisation mit hochauflösenden Massenspektrometrie gekoppelt

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Synthese- und Massenspektrometrie der Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

Charakterisierung von synthetischen Polymeren Matrix Assisted Laser Desorption Ionisation Zeit der Massenspektrometrie Flug (MALDI-TOF)

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

Eine effiziente Vorbereitung Beispielmethode Kohlenhydrat Ionen-Signale in der Matrix-unterstützte Laser Desorption/Ionisierung Massenspektrometrie verbessern

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Analyse komplexer Moleküle und ihrer Reaktionen auf Oberflächen mittels Cluster-induzierter Desorption/Ionisationsmassspektrometrie

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

Kapillarelektrophorese Massenspektrometrie Ansätze zur Charakterisierung der protein- und metaboliten Korona, die von Nanomaterialien erfasst werden

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

Kovalente Markierung mit Diethylpyrocarbonat zur Untersuchung der Proteinstruktur höherer Ordnung durch Massenspektrometrie

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Organic Solvent-Based Protein Precipitation for Robust Proteome Purification Ahead of Mass Spectrometry

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end approaches to isolate, enrich, and process proteins ahead of MS are critical for successful proteome characterization. Low, inconsistent protein recovery and residual impurities such as surfactants are detrimental to MS analysis. Protein precipitation is often considered unreliable, time-consuming, and technically challenging to perform compared to other sample preparation strategies. These concerns are overcome by employing optimal protein precipitation protocols. For acetone precipitation, the combination of specific salts, temperature control, solvent composition, and precipitation time is critical, while the efficiency of chloroform/methanol/water precipitation depends on proper pipetting and vial manipulation. Alternatively, these precipitation protocols are streamlined and semi-automated within a disposable spin cartridge. The expected outcomes of solvent-based protein precipitation in the conventional format and using a disposable, two-stage filtration and extraction cartridge are illustrated in this work. This includes the detailed characterization of proteomic mixtures by bottom-up LC-MS/MS analysis. The superior performance of SDS-based workflows is also demonstrated relative to non-contaminated protein.

The present protocol describes solvent-based protein precipitation under controlled conditions for robust and rapid recovery and purification of proteome samples prior to mass spectrometry.

Proteinfällung auf organischer Lösungsmittelbasis für eine robuste Proteomreinigung vor der Massenspektrometrie

While multiple advances in mass spectrometry (MS) instruments have improved qualitative and quantitative proteome analysis, more reliable front-end ...