Name: A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry (Video) | JoVE Journal
Uploaded: 2016-01-08T13:00:01.000+00:00
Duration: 12 min 59 s
Description: Learn to characterize mercury-dicysteinyl peptide complexes using electrospray ionization orbitrap mass spectrometry. This method enables analysis with minimal fragmentation and distinct isotopic patterns for designing chelating agents.

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5 minutes

M.N-S acknowledges support from the National Science Foundation, RUI grant CHE 1011859. The authors gratefully acknowledge the Triad Mass Spectrometry Facility at the University of North Carolina at Greensboro for use of the Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer. The authors thank Daniel Todd, Vincent Sica, and Brandie Erhmann at the University of North Carolina at Greensboro for helpful suggestions and comments regarding this work.

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
<ol>
	<li>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 water to the calibration line to make a 5 mM ammonium formate solution.</li>
	<li>Degas the 5 mM ammonium formate buffer under a vacuum system for 10 min and purge with argon. Repeat twice and store solution under argon. On the day of use, filter the buffer solution through a 0.2 micron filter before use.</li>
</ol>
2. Preparation of Mercury(II) Chloride Solutions
<ol>
	<li>Weigh out 0.2375 g mercury(II) chloride. Dissolve it in 25 ml of 5 mM ammonium formate buffer to produce a 0.035 M mercury(II) chloride solution.</li>
	<li>Add 0.214 ml of 0.035 M mercury(II) chloride solution to 9.785 ml of 5 mM ammonium formate buffer to create a 7.5 x 10-4 M solution. Blanket the 7.5 x 10-4 M mercury(II) solution with argon gas.</li>
</ol>
3. Preparation of CGGC Stock Solution
<ol>
	<li>Dissolve 2.0 mg of the dicysteinyl tetrapeptide, CGGC, in 0.118 ml of HPLC grade acetonitrile and then add 1.0647 ml of 5 mM ammonium formate, pH 7.5 buffer that has been degassed in argon to yield a 5 mM CGGC stock solution.</li>
	<li>Add 225 &#181;l of the 5 mM CGGC stock solution to 1,275 &#181;l of 5 mM ammonium formation pH 7.5 buffer to give a 7.5 x 10-4 M CGGC solution.</li>
</ol>
4. Preparation of Various Reaction Mixtures of Mercury(II) and CGGC
<ol>
	<li>Preparation of 1:0.5 ratio of mercury(II):CGGC solution
	<ol>
		<li>Place 255 &#181;l of 5 mM ammonium formate, pH 7.5 buffer into a 1.5 ml microcentrifuge tube. Add 30 &#181;l of 7.5 x10-4 M mercury(II) chloride solution into the 1.5 ml microcentrifuge tube with the ammonium formate buffer.</li>
		<li>Vortex the solution for 10 sec. Then add 15 &#181;l of 7.5 x 10-4 M CGGC solution into the 1.5 ml microcentrifuge. Vortex the solution for 10 sec. Let the solution stand for 10 min&#160;prior to injection into the mass spectrometer.</li>
	</ol>
	</li>
	<li>Preparation of 1:1 ratio of mercury(II):CGGC solution
	<ol>
		<li>Place 240 &#181;l of 5 mM ammonium formate, pH 7.5 buffer into a 1.5 ml microcentrifuge tube. Add 30 &#181;l of 7.5 x10-4 M mercury(II) chloride solution into the 1.5 ml microcentrifuge tube with the ammonium formate buffer.</li>
		<li>Vortex the solution for 10 sec. Then add 30 &#181;l of 7.5 x 10 -4 M CGGC solution into the 1.5 ml microcentrifuge tube. Repeat in a similar manner as described in section 4.1.</li>
	</ol>
	</li>
	<li>Preparation of 1:2 ratio of mercury(II):CGGC solution
	<ol>
		<li>Place 210 &#181;l of 5 mM ammonium formate, pH 7.5 buffer into a 1.5 ml microcentrifuge tube. Add 30 &#181;l of 7.5 x10-4 M mercury(II) chloride solution into the 1.5 ml microcentrifuge tube with the ammonium formate buffer.</li>
		<li>Vortex the solution for 10 sec. Then add 60 &#181;l of 7.5 x 10-4 M CGGC solution into the 1.5 ml microcentrifuge tube. Repeat in a similar manner as described in section 4.1.</li>
	</ol>
	</li>
</ol>
5. Preparation of CEEC Stock Solution
<ol>
	<li>Dissolve 3.5 mg of the dicysteinyl tetrapeptide, CEEC, in 0.145 ml of HPLC grade acetonitrile to dissolve the peptide. Then add 13.067 ml of 5 mM ammonium formate, pH 7.5 buffer that has been degassed in argon to produce 0.5 mM CEEC solution.</li>
	<li>Vortex the solution until all peptide is dissolved. Add 1.125 ml of 0.5 mM CEEC solution and 0.375 ml of 5 mM ammonium formate, pH 7.5 buffer to a 1.5 ml microcentrifuge tube to give a 7.5 x 10-5 M CEEC solution. Vortex until mixed.</li>
</ol>
6. Preparation of Various Reaction Mixtures of Mercury(II) and CEEC Solution
<ol>
	<li>Preparation of 1:0.5 ratio of mercury(II):CEEC solution
	<ol>
		<li>Place 255 &#181;l of 5 mM ammonium formate, pH 7.5 buffer into a 1.5 ml microcentrifuge tube. Add 30 &#181;l of 7.5 x10-4 M mercury(II) chloride solution into the 1.5 ml microcentrifuge tube with the ammonium formate buffer.</li>
		<li>Vortex the solution for 10 sec. Then add 15 &#181;l of 7.5 x 10-4 M CEEC solution into the 1.5 ml microcentrifuge tube. Repeat in a similar manner as described in section 4.1.</li>
	</ol>
	</li>
	<li>Preparation of 1:1 ratio of mercury(II):CEEC solution
	<ol>
		<li>Place 240 &#181;l of 5 mM ammonium formate, pH 7.5 buffer into a 1.5 ml microcentrifuge tube. Add 30 &#181;l of 7.5 x10-4 M mercury(II) chloride solution into the 1.5 ml microcentrifuge tube with the ammonium formate buffer.</li>
		<li>Vortex the solution for 10 sec. Then add 30 &#181;l of 7.5 x 10-4 M CEEC solution into the 1.5 ml microcentrifuge tube. Repeat in a similar manner as described in section 4.1.</li>
	</ol>
	</li>
	<li>Preparation of 1:2 ratio of mercury(II):CEEC solution
	<ol>
		<li>Place 210 &#181;l of 5 mM ammonium formate, pH 7.5 buffer into a 1.5 ml microcentrifuge tube. Add 30 &#181;l of 7.5 x10-4 M mercury(II) chloride solution into the 1.5 ml microcentrifuge tube with the ammonium formate buffer.</li>
		<li>Vortex the solution for 10 sec. Then add 60 &#181;l of 7.5 x 10-4 M CEEC solution into the 1.5 ml microcentrifuge tube. Repeat in a similar manner as described in section 4.1.</li>
	</ol>
	</li>
</ol>
7. Analyzing the Reaction Mixtures of Mercury(II) and CGGC Samples by Orbitrap ESI Mass Spectrometry
<ol>
	<li>Preparing the ESI mass spectrometer16
	<ol>
		<li>Draw 100 &#181;l of calibration standards into a 500 &#181;l glass syringe.</li>
		<li>Place the syringe in the syringe cradle of the MS pump, attach the tubing, and inject into the mass spectrophotometer.</li>
		<li>Set up the file name for the run by selecting the file icon and typing the file name.</li>
		<li>Select acquire data button in the data acquisition module and collect 150 scans.</li>
		<li>Analyze the chromatogram to verify the calibration standards by opening data processing module of the software. Open the module, go to file menu and select &#34;open&#34;, and select the file in the dialog box. Verify that the peaks in the chromatogram correlate to the mass to charge ratios of the standards.</li>
		<li>Clean the 500 &#181;l glass syringe by drawing up 500 &#181;l HPLC grade methanol and then dispense the methanol into a beaker.</li>
		<li>Draw up 500 &#181;l of HPLC grade methanol into the glass syringe and flush the system as per step 7.1.2.</li>
		<li>Select the method setup module of the software to set the parameters. Choose the scan mode menu and identify the analyzer as FTMS, and then click on &#34;OK&#34;. Then by clicking on the various icons on the real-time view spectrum page, set the following parameters: Sheath gas flow rate: 10, Source temperature: 0, Capillary voltage: 37 V, Tube lens: 95 V, Spray voltage: 4.20 kV, Flow rate 10.00 &#181;l/min, Analyzer: FTMS, Number of scans: 150.</li>
	</ol>
	</li>
	<li>Running CGGC samples on ESI mass spectrometer
	<ol>
		<li>Run the 5 mM ammonium formate pH 7.5 buffer.
		<ol>
			<li>Place 500 &#181;l of 5 mM ammonium formate buffer into the 500 &#181;l glass syringe, place it into the syringe cradle of the MS pump, and attach the tubing.</li>
			<li>Run buffer through the tubing for 1-2 min.</li>
			<li>Set up the file name for the run by selecting the file icon and typing the file name.</li>
			<li>Select acquire data button in the module and collect 150 scans.</li>
			<li>Click the run button to stop collection after 150 scans are collected.</li>
			<li>Open the data browser module, then go to file menu and select &#34;open&#34;, and select the file in the dialog box. Verify that no peaks at 483, 683, 1,163 and 1,363 m/z are present that resemble the peptide or mercury (II)-peptide complexes.</li>
		</ol>
		</li>
		<li>Run the 1:0.5 mercury(II):CGGC ratio solution.
		<ol>
			<li>Place 250 &#181;l of 1:0.5 mercury(II):CGGC ratio of the sample into the syringe.</li>
			<li>Place the syringe into the syringe cradle of the MS pump, attach the tubing, and prime the apparatus.</li>
			<li>Select a file name for the run by selecting the file icon and typing the file name.</li>
			<li>Press the acquire data button in the data acquisition module and collect 150 scans and click the run button to stop the collection.</li>
			<li>Open the module, go to file menu and select &#34;open&#34;, and select the file in the dialog box. Verify that the chromatogram contains peaks including the one for the CGGC peptide alone.</li>
			<li>Wash the syringe by aspirating with 500 &#181;l ammonium formate buffer and then dispensing the ammonium formate buffer into a beaker.</li>
			<li>Select the waste button on the MS and flush the tubing three times with 500 &#181;l ammonium formate buffer.</li>
			<li>Wash the syringe by aspirating with 500 &#181;l methanol and then dispensing the methanol into a beaker.</li>
			<li>Flush the tubing one time with 500 &#181;l methanol.</li>
			<li>Select load detector button on the MS.</li>
			<li>Add 500 &#181;l of ammonium formate buffer to the syringe.</li>
			<li>Place the syringe into the syringe cradle of the MS pump, attach the tubing, and prime the apparatus.</li>
			<li>Select a file name for the buffer run by selecting the file icon and typing the file name.</li>
			<li>Press the acquire data button and collect 150 scans and then click the stop run button.</li>
			<li>Open the data browser module, go to file menu and select &#34;open&#34;, and select the file in the dialog box. Verify that the chromatogram is void of peaks from the previous Hg:CGGC run.</li>
		</ol>
		</li>
		<li>Run the 1:1 mercury(II):CGGC ratio solution.
		<ol>
			<li>Place 250 &#181;l of 1:1 mercury(II):CGGC ratio sample into the syringe.</li>
			<li>Repeat in a similar manner as described for Steps 7.2.2.2 to 7.2.2.15.</li>
		</ol>
		</li>
		<li>Run the 1:2 mercury(II):CGGC ratio solution
		<ol>
			<li>Place 250 &#181;l of 1:2 mercury(II):CGGC concentration sample into the syringe.</li>
			<li>Repeat in a similar manner as described for Steps 7.2.2.2 to 7.2.2.15.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
8. Analyzing the Reaction Mixtures of Mercury and CEEC Samples by Orbitrap ESI Mass Spectrometry
<ol>
	<li>Running CEEC samples on ESI mass spectrometer
	<ol>
		<li>Repeat analysis procedure (Steps 7.1 to 7.2) using CEEC samples and reaction mixtures of mercury(II) and CEEC at various stoichiometric ratios.</li>
	</ol>
	</li>
</ol>

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There is no disclosure for the work reported.

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...

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><tbody><tr><td>Mercury(II) chloride</td><td>Sigma-Aldrich</td><td>429724</td><td>Highly toxic</td></tr><tr><td>Ammonium formate</td><td>Sigma-Aldrich</td><td>516961</td><td></td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>Ammonium hydroxide</td><td>Fisher</td><td>A512-P500</td><td></td></tr><tr><td>HPLC water</td><td>Fisher</td><td>W5-4</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Fisher</td><td>BP2405-1</td><td></td></tr><tr><td>HPLC Methanol</td><td>Fisher</td><td>A452-4</td><td></td></tr></tbody></table>

a study of the complexation of mercury(ii) with dicysteinyl tetrapeptides by electrospray ionization mass spectrometry

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.

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.
...

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

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

The overall goal of this procedure is to prepare complexes of mercury and dicysteinyl peptides under different reaction stoichiometries, and to characterize the different complexes formed by electrospray ionization and oribitrap mass spectrometry. This method can help answer key questions regarding the complexation tendencies of mercury and thiol compounds, which will enhance the rational design of chelating agents for sequestering mercury ions. The main advantage of this technique is that it facilitates the analysis of metal ion complexes with negligible fragmentation.Others include distinct mercury isotopic distribution pattern. Ease in assigning the charge and analyzing overlapping peaks by tandem mass spectrometry. Though this method provides insight into mercury peptide complexes, it can also be applied with other complexes, especially with metal ions that exist in different isotopic forms.Degas a five millimolar ammonium formate buffer under a vacuum system for 10 minutes, and purge with argon. Repeat twice and store the solution under argon. On the day of use, filter the buffer solution through a 0.2 micron filter before use.To prepare mercury chloride solutions, weigh out 0.2375 grams of mercury chloride. Dissolve in 25 milliliters of five millimolar ammonium formate buffer, to produce a 0.035 molar mercury chloride solution. Add 0.214 milliliters of the mercury chloride solution to 9.785 milliliters of five millimolar ammonium formate buffer, to create a 0.75 millimolar solution.Blanket the resulting solution with argon gas. Next, add 225 microliters of a five millimolar CGGC stock solution to 1.275 milliliters of five millimolar ammonium formate pH 7.5 buffer, to give a 0.75 millimolar CGGC solution. Then, prepare a one to 0.5 ratio of mercury to CGGC solution by placing 255 microliters of five millimolar ammonium formate pH 7.5 buffer into a 1.5 milliliter microcentrifuge tube.Then, add 30 microliters of 0.75 millimolar mercury chloride solution into the tube. Vortex the solution for 10 seconds. Then, add 15 microliters of 0.75 millimolar CGGC solution into the tube.Vortex the solution for another 10 seconds, and then let the solution stand for 10 minutes prior to injection into the mass spectrometer. To prepare the ESI mass spectrometer, or MS, draw 100 microliters of calibration standards into a 500 microliter glass syringe. Place the syringe in the syringe cradle of the MS pump.Attach the tubing, and inject into the mass spectrometer. Set up the file name for the run by selecting the file icon and typing the file name. Select the acquire data button in the data acquisition module, and collect 150 scans.Analyze the chromatogram to verify the calibration standards by opening the data processing module of the software. Then, go to the file menu, and select open, followed by the file in the dialogue box. Verify that the peaks in the chromatogram correlate with the mass to charge ratios of the standards.Clean the 500 microliter glass syringe by drawing up 500 microliters of HPLC grade methanol and then dispensing it into a beaker. Draw up 500 microliters of HPLC grade methanol into the glass syringe, and flush the system. Select the method setup module of the software to set the parameters.Choose the scan mode menu and identify the analyzers FTMS, then click on okay. Next, click through the various icons on the real time view spectrum page to set up the parameters as detailed in the text protocol. To run the buffer, place 500 microliters of five millimolar ammonium formate buffer into the 500 microliter glass syringe.Place it into the syringe cradle of the MS pump, and detach the tubing. Run the buffer through the tubing for one to two minutes. Set up the file name for the run, by selecting the file icon and typing the file name.Select the acquire data button in the module, and collect 150 scans. Click the stop run button to stop collection after 150 scans are collected. Next, open the data browser module, go to file menu and select open, followed by the file in the dialogue box.Verify that no peaks are present at 483, 683, 1163, and 1363 mass overcharge that resemble the peptide or mercury ion peptide complexes. Run the one to 0.5 mercury to CGGC ratio solution by placing 250 microliters of the sample into the syringe. Place the syringe into the syringe cradle of the MS pump.Attach the tubing, and prime the apparatus. Acquiring 150 data scans, open the browser module, go to file menu, and select open. Then, select the file in the dialogue box.Verify that the chromatogram contains peaks including the one for the CGGC peptide alone. A critical aspect of this procedure is to ensure that analytes from the previous run are completely removed prior to each reaction mixture analysis. Minimization of sample contamination from carryover analytes is ensured by cleaning the injection line thoroughly.Wash the syringe by aspirating with 500 microliters of ammonium formate buffer, and then dispensing the buffer into a beaker. Then, select the waste button on the MS and flush the tubing three times with 500 microliters of ammonium formate buffer. Further wash the syringe, by aspirating 500 microliters of methanol and dispensing into a beaker.Flush the tubing one time with 500 microliters of methanol. Then, select the load detector button on the MS.After adding 500 microliters of ammonium formate buffer to the syringe, place the syringe into the syringe cradle of the MS pump. Attach the tubing, and prime the apparatus.Select a file name for the buffer run as before, and acquire 150 scans before clicking the stop run button. Open the data browser module to verify that the chromatogram is void of peaks from the previous mercury to CGGC run. Repeat the analysis procedure for the remaining CGGC samples.To prepare a one to 0.5 ratio of mercury to the CEEC solution, place 255 microliters of five millimolar ammonium formate pH 7.5 buffer, into a 1.5 milliliter microcentrifuge tube. Add 30 microliters of 0.75 millimolar mercury chloride solution into the tube. After vortexing the solution for 10 seconds, add 15 microliters of a 0.75 millimolar CEEC solution into the tube.Vortex the solution for another 10 seconds. And let the solution stand for 10 minutes prior to mass spectrometer injection. Repeat the analysis procedure using CEEC samples and reaction mixtures of mercury ion and CEEC.A representative mass spectrum is shown. After 10 minutes, the various types of mercury peptide complexes are visible for the one to 0.5 mercury to CGGC reaction mix, along with a peptide peak of 339.08, and peptide dimer of 677.15. As observed in the prior spectrum, the one to one mercury to CGGC reaction mixture contains some similar peaks.For example, peak 539.03 indicates the one to one complex of mercury and peptide. The zoomed-in view exhibits the signature mercury isotopic pattern corresponding to the seven main naturally occurring mercury isotopes. Also observed, is a one to two mercury to CGGC complex, as previously reported for mercury dicysteinyl tripeptide.This complex appears in all three reaction mixtures of mercury and CGGC. It was readily analyzed by a distinct mercury isotopic pattern with a monoisotopic peak at 877. However, if the mercury is in excess, as in the one to 0.5 mercury to CGGC reaction mixture, an additional two to two mercury to CGGC complex, is detected.This mercury isotopic signature corresponds to a two mercury complex as calculated by using the chem cal program. The theoretical protonated monoisotopic mass corresponds to a mass overcharge value of 1077, which is the ninth peak in the observed isotopic cluster. A representative spectrum showing various mercury to CEEC complexes, is shown.Of interest, is the overlapping peaks associated with mercury to CEEC adducts in the plus one charge showing a monoisotopic peak at 883. And in the plus two charge, with a calculated monoisotopic peak value of 1765. Once mastered, this technique can be done in less than two hours, if it is performed properly.While attempting this procedure, it's important to remember to degas your buffers and rinse the line thoroughly to avoid carryover analytes. Following this procedure, other methods like proton, carbon-13 and mercury-199 nuclear magnetic resonance spectroscopy, or potentiometry, can be utilized to provide a more accurate determination of the content complexes in the solution phase. After watching this video, you should be able to analyze various mercury complexes and their distinct mercury isotopic distribution patterns and charge formations using orbitrap mass spectrometry with electrospray ionization, as well as to distinguish overlapping peaks by tandem MS analysis.Don't forget that working with mercury solutions can be extremely hazardous and precaution such as wearing personal protective equipment should always be taken while performing this procedure.

a-study-complexation-mercury-ii-with-dicysteinyl-tetrapeptides

Research

JoVE Journal

Chemistry

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

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