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.
