Ion mobility-mass spectrometry, or IM-MS, identify the various products ions from the pH-dependent redox and methyl binding reaction of peptides. With the molecular modeling of their tertiary structure, metal correlation can be determined. IM-MS can resolve each of the product ions and identify their molecular composition by simultaneously measuring their mass-to-charge and arrival times and relating to their stoichiometry, protonation state, and conformational structure.
Developing classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance, such as Menkes and Wilson's disease, cancers, and Alzheimer's disease. To begin, clean the ESI entrance tubing and needle capillary thoroughly with about 500 microliters of 0.1 molar glacial acetic acid, 0.1 molar ammonium hydroxide, and finally, deionized water. Use native ESI-IM-MS conditions as described in the text protocol to collect the negative and positive ion IM-MS spectra of the 10 ppm poly-DL-alanine solution for 10 minutes each.
Pipette 200.0 microliters of the 0.125 millimolar alternative methanobactin, or amb solution, into a 1.7 milliliter vial. Dilute with 500 microliters of deionized water and mix the solution thoroughly. Adjust the pH of the sample to 3.0 by adding 50 microliters of 1.0 molar acetic acid solution.
Add 200.0 microliters of the 0.125 millimolar metal ion to the pH-adjusted sample. Then, add deionized water to yield a final volume of 1.00 milliliter of the sample. Mix thoroughly, and allow the sample to equilibrate for 10 minutes at room temperature.
Using a blunt nose syringe, take 500 microliters of the sample and collect the negative and positive ion ESI-IM-MS spectra for five minutes each. Use the remaining 500 microliters of the sample to record its final pH using a calibrated micro pH electrode. Repeat these steps, except to adjust the pH to pH four, five, six, seven, eight, nine, or 10, by adding new volumes of acetic acid or ammonium hydroxide solutions.
Collect the negative and positive ion ESI-IM-MS spectra of the resulting solutions for 10 minutes each. From the IM-MS spectra, identify which charged species of the alternative methanobactin are present by matching them to their theoretical mass-to-charge isotope patterns. To do so, open MassLynx and click on Chromatogram to open the Chromatogram window.
Go to the File menu and Open to locate and open the IM-MS data file. Extract the IM-MS spectrum by right-clicking, dragging across the chromatogram, and releasing. The spectrum window will open, showing the IM-MS spectrum.
In the spectrum window, click on Tools and Isotope Model. In the Isotope modeling window, enter the molecular formula of the amb species, check the Show charged ion box, and enter the charge state. Click OK.Repeat this process to identify all of the species in the IM-MS spectrum, and record their mass-to-charge isotope range.
For each amb species, separate any coincidental mass-to-charge species and extract their arrival time distributions, or ATDs, using their mass-to-charge isotope patterns to identify them. Open DriftScope and click on File and Open to locate and open the IM-MS data file. Use the mouse and left-click to zoom in on the mass-to-charge isotope pattern of the amb species.
Use the Selection Tool and left mouse button to select the isotope pattern. Click the Accept current selection button. To separate any coincidental mass-to-charge species, use the Selection Tool and left mouse button to select the ATD time aligned with isotope pattern of the amb species.
Click the Accept current selection button. To export the ATD, go to File, Export to MassLynx. Then select Retain Drift Time and save the file to the appropriate folder.
In the Chromatogram window of MassLynx, open the saved exported file. Click on Process, Integrate from the menu, check the ApexTrack Peak Integration box, and click OK.Record the centroid and the integrated area of the ATD. After repeating this process for all saved amb and poly-DL-alanine IM-MS data files, use the integrated ATD for all extracted amb species of either the positive or the negative ions at each titration point to normalize to a relative percentage scale.
To do so, enter the identities of the amb species and their integrated ATD at each pH into a spreadsheet. For each pH, use the sum of the integrated ATDs to normalize the individual amb species'ATD to a percentage scale. Plot the percent intensities of each amb species versus pH to show how the population of each species varies as a function of pH.
Using a spreadsheet, convert the collision cross sections of poly-DL-alanine negative and positive ions measured in helium buffer gas to the corrected collision cross sections. Then, convert the average arrival times of the poly-DL-alanine calibrants and amb species into drift times. Plug the poly-DL-alanine calibrants'drift times versus their corrected collision cross sections.
Then, using a least squares regression fit, determine the A prime and B values, where A prime is the correction for the temperature, pressure, and electric field parameters, and B compensates for the nonlinear effect of the IM device. Using these A prime and B values with the centroid drift times value, determine their corrected collision cross sections and their collision cross sections. This method provides collision cross sections for the peptide species with estimated absolute errors of about 2%Using Gaussian with GaussView, and the B3LYP LanL2DZ level of theory, locate geometry-optimized conformers for all possible types of coordinations of the observed mass-to-charge amb species.
The B3LYP LanL2DZ level of theory is comprised of the Becke three perimeter hybrid functionals, the Dunning basis set, and electron core potentials. Extract the thermochemical analyses of each of the optimized conformers from the Gaussian output file and calculate their theoretical collision cross sections using the ion-scaled Lennard-Jones method from the Sigma program. From the lowest free energy conformers, determine which conformer exhibits the Lennard-Jones collision cross section that agrees with the IM-MS measured collision cross section.
This process identifies the tertiary structure and type of coordination for the conformers observed in the experiment. Molecular modeling requires comparison of the free energy and collision cross sections of conformers with different metal chelating sites, cis and trans peptide bonds, salt bridges, hydrogen bonding, and pi-cation interactions. The IM-MS study of the alternative methanobactin one showed that it chelated both copper and zinc ions in a pH-dependent manner, but through different reaction mechanisms and coordination sites.
Zinc(II)binding was observed at a pH of greater than six, primarily forming a single negatively charged complex, indicating the zinc(II)was tetrahedrally coordinated by the two imidazoles and two thiolates. Copper(II)binding was accompanied by the thiols forming a disulfide bridge. At a pH of greater than six, the single negatively charged copper(II)complex was formed, indicating imidazole and two deprotonated amide nitrogens were coordinating copper(II)However, below pH six, adding copper(II)also formed the single positively charged copper(I)complex, as well as the single positively charged copper(II)complex above pH six.
IM-MS studies of amb two and amb four also show that the copper reactions gave products that differed in the numbers of inter-or intramolecular disulfide bridges, number of copper(I)or copper(II)ions, and number of deprotonation sites, which changed as a function of pH. The IM-MS result with molecular modeling showed that the alternative methanobactins could coordinate up to three copper(I)ions via the thiolate, imidazoles, and carboxylate groups. The IM-MS instrumental settings must be carefully chosen to conserve the peptides'stoichiometry, charge distributions, and conformational structures, as described in the text.
Combination of wider range of size that will influence metal coordination such as the tyrosine at aspartic acid will allow a greater understanding of the relationship between structure and function. IM-MS with molecular modeling have become alternative techniques to accelerate crystallography and NMR spectroscopy for determining the conformational structures of proteins, DNA, lipids, and their complexes.
