Uno studio della Complessazione di Mercurio (II) con Dicysteinyl Tetrapeptidi da Electrospray ionizzazione Spettrometria di Massa
Summary
The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
Il dicysteinyl tetrapeptide idrofoba CGGC (C 10 H 18 N 4 O 5 S 2; PM = 338) (Figura 1), forma complessi con mercurio (II) come mostrato de Figura 2 e Tabella 1 Inoltre, forma dimeri peptidici e trimeri. incrementale come aumenta la quantità di peptide nella miscela di reazione. Come mostrato dai valori m / z dei dimeri associati [(2M + H) + = 677] e trimeri [(3M + H) + = 1.015…
Declarações
The authors have nothing to disclose.
Acknowledgements
MN-S riconosce il sostegno della National Science Foundation, RUI concedere CHE 1011859. Gli autori ringraziano lo strumento Triade Spettrometria di Massa presso la University of North Carolina a Greensboro per l'uso del spettrometro di massa Thermo Fisher Scientific LTQ Orbitrap XL. Gli autori ringraziano Daniel Todd, Vincenzo Sica, e Brandie Erhmann presso la University of North Carolina a Greensboro per utili suggerimenti e commenti questo lavoro.
Materials
| Mercury(II) chloride | Sigma-Aldrich | 429724 | Highly toxic |
| Ammonium formate | Sigma-Aldrich | 516961 | |
| Formic acid | Sigma-Aldrich | F0507 | |
| Ammonium hydroxide | Fisher | A512-P500 | |
| HPLC water | Fisher | W5-4 | |
| HPLC Acetonitrile | Fisher | BP2405-1 | |
| HPLC Methanol | Fisher | A452-4 |
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