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

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 ions^2,3. However, studies have shown that these small thiol compounds [dimercaptosuccinic acid (DMSA) and dimercaptopropane-sulfonic acid (DMPS)] are not optimal for mercury chelation therapy^4-6. 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....

Protocol

Note: Please consult all relevant material safety data sheets (MSDS) before use. Mercury chloride is a toxic chemical. Personal protective equipment (gloves, safety goggles, and lab coat) must be worn when handing it and all associated solutions. Dispose of solutions in clearly labeled chemical waste bottles designated for heavy metals.

1. Preparation of 5 mM Degassed Ammonium Formate Buffer, pH 7.5

1. Dissolve 0.1576 g of ammonium formate buffer in 450 ml of HPLC grade water. Adjust the pH of the above solution with 1 M formic acid and 1 M ammonium hydroxide to 7.5. Transfer this solution to a 500 ml volumetric flask and add HPLC ....

Results

A study was performed to characterize the possible mercury-peptide complex composition for two tetrapeptides, CGGC and CEEC (Figure 1) by ESI mass spectrometry. Complexes of mercury(II) with CGGC or CEEC were investigated by reacting the mixtures of mercury(II) and peptide solutions at three different molar ratios: 1:0.5, 1:1, and 1:2 (mercury(II): peptide). The concentration of mercury(II) was 7.5 x 10^-6 M and the peptide concentration varied accordingly.

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Discussion

The hydrophobic dicysteinyl tetrapeptide CGGC (C₁₀H₁₈N₄O₅S₂; MW = 338) (Figure 1), forms complexes with mercury(II) as shown in Figure 2 and Table 1. Additionally, it forms peptide dimers and trimers incrementally as the amount of peptide increases in the reaction mixture. As shown by the m/z values of the associated dimers [(2M+H)⁺ = 677] and trimers [(3M+H)⁺ = 1015], the thiol groups of CGGC d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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