Name: a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry
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Description: Watch this Scientific Journal Video about A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry at JoVE.com

MN-S erkent steun van de National Science Foundation, RUI verlenen CHE 1011859. De auteurs dankbaar erkennen de Triade Massaspectrometrie Facility aan de Universiteit van North Carolina in Greensboro voor het gebruik van de Thermo Fisher Scientific LTQ Orbitrap XL massaspectrometer. De auteurs danken Daniel Todd, Vincent Sica en Brandie Erhmann aan de Universiteit van North Carolina in Greensboro voor nuttige suggesties en opmerkingen met betrekking tot dit werk.

Opmerking: alle relevante veiligheidsinformatiebladen (VIB) te raadplegen voor gebruik. Kwik chloride is een giftige chemische stof. Persoonlijke beschermingsmiddelen (handschoenen, veiligheidsbril en laboratoriumjas) moet gedragen worden bij af te voeren en alle bijbehorende oplossingen. Gooi oplossingen duidelijk gelabeld chemisch afval flessen aangewezen voor zware metalen. 1. Bereiding van 5 mM Ontgaste ammoniumformaatbuffer, pH 7,5 <ol><li> Oplossen in 450 ml HPLC kwaliteit water 0,15...

<ol>
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De hydrofobe dicysteinyl tetrapeptide CGGC (C 10 H 18 N 4 O 5 S 2, MW = 338) (figuur 1) vormt complexen met kwik (II) zoals in figuur 2 en tabel 1 Daarnaast vormt het peptide dimeren en trimeren. stapsgewijs de hoeveelheid peptide toename in het reactiemengsel. Zoals blijkt uit de m / z-waarden van de bijbehorende dimeren [(2M + H) + = 677] en trimeren [(3M + H) + = 1015], de t...

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<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> Een onderzoek werd uitgevoerd om de mogelijke kwik-peptidecomplex karakteriseren preparaat twee tetrapeptiden, CGGC en LMOE (figuur 1) van ESI massaspectrometrie. Complexen kwik (II) met CGGC of LMOE werden onderzocht door het laten reageren van het mengsel van kwik (II) en peptideoplossingen drie verschillende molaire verhoudingen: 1: 0,5, 1: 1 en 1: 2 (kwik (II): peptide) . De concentratie van kwik (II) was 7,5 x 10 -6 M en peptideconcentratie dienover...

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.

Een studie van de Complexatie van Mercury (II) met Dicysteinyl tetrapeptiden door electrospray ionisatie massaspectrometrie

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

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

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