Elektrosprey İyonizasyon kütle spektrometresi ile Dicysteinyl tetrapeptidlerle ile Merkür (II) kompleksinin Bir Çalışma
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
Hidrofobik dicysteinyl tetrapeptit CGGC (Cı 10H 18 N 4 O 5S 2 MW = 338) (Şekil 1), cıva kompleksler oluşturan (II), Şekil 2'de gösterilen ve Tablo 1 Ayrıca, peptit dimerler ve trimerler de oluşturmasıyla ayrılır. aşamalı olarak, reaksiyon karışımı içindeki peptit miktarı arttıkça. M / z ilişkili dimerlerin değerleri [(2M + H) + = 677] ve trimerl…
Declarações
The authors have nothing to disclose.
Acknowledgements
MN-S Ulusal Bilim Vakfı desteği kabul RUI yazarları minnetle Thermo Fisher Scientific LTQ Orbitrap XL kütle spektrometresi kullanımına ilişkin Greensboro, Kuzey Carolina Üniversitesi'nde Triad Kütle Spektrometre Tesisi kabul Che 1011859. verin. Yazarlar bu çalışmaları ile ilgili faydalı görüş ve önerileriniz için Greensboro, Kuzey Carolina Üniversitesi'nden Daniel Todd, Vincent Sica ve Brandie Erhmann teşekkür ederiz.
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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