This new technique combines ion mobility-mass spectrometry with molecular modeling and reaction dynamics theory to determine the relative thermochemistry of two competing dissociation reactions of a ternary complex. The combination of these techniques characterizes the reactants and products reaction pathways, conformational structures, and the ligand's affinity to form a ternary complex with the metal ion. This research models the reactivity of two potential peptide tags for recombinant protein purification, where the tag reacts with the metal chelated by nitrilotriacetic acid in an immobilized metal affinity column.
Begin by loading the 2 milliliter sample into a 2.5-milliliter blunt nose syringe and injecting the sample into the electrospray of the instrument using the instrument's syringe pump at a flow rate of 10 microliters per minute. Place the instrument in negative ion mobility-mass spectrometry, or IM-MS mode. Identify the mass-to-charge isotope pattern of the negatively-charged alternative metal binding ternary complex, or amb-metal-NTA complex, by opening the mass spectrometry program and selecting Spectrum.
Then, select Tools followed by Isotope Model. In the popup window, list the molecular formula of the complex, check the box for Show Charged Ion, enter 1 for the charge of negative one, and click on OK.In the displayed isotope pattern of the complex, note the lowest mass peak. In the instrument software, select Setup followed by Quad Profile.
Then, select Manual Fixed and enter the mass of the lowest isotopic pattern peak. Click on Update and close the window. Again, select Setup, and then click on Resolving Quad.
Collect the negative ion IM-MS spectra starting with the first transfer collision energy using a 5-minute run duration and 2-second scan time. Repeat to collect the IM-MS spectra for each of the other collision energies. Utilize the integrated arrival time distributions, or ATD areas, for the ternary amb-metal-NTA complex and two products, NTA-metal complex and amb-metal complex, to normalize to a relative percentage scale.
From the replicate threshold collision-induced dissociation, or TCID measurements, find the mean and standard deviations of each data point. Then, convert the lab-frame transfer collision energy to the center-of-mass collision energy. To measure collision cross-sections, collect the negative ion IM-MS spectra of the 10 ppm poly-DL-alanine, or PA sample, for 10 minutes using the instrumental operating conditions.
Then, collect the IM-MS spectra of each ternary complex for 5 minutes. Extract the ATD of each of the PA and ternary complexes and export their files to the mass spectrometry software using the Retain Drift Time option. Find the average arrival times from the maxima of the corresponding ATD curves.
Use a cross-sectional calibration method to convert the average arrival times to collision cross-sections of the ternary complex. From the CRUNCH main menu, open the GB5 text file containing the energy of the center-of-mass, or ECM-dependent relative intensities of the products. Reply No to read parameters.
Then, select Modeling followed by Set All Parameters. From the reaction model options, choose the default Threshold CID option followed by RRKM with integration over the energy transfer distribution of the ternary complex. Enter 2 for independent product channels modeled, and select Calculate cross sections.
For the type of unconvoluted model, choose 0 Kelvin cross section, which includes the statistical RRKM correction of the kinetic shifts due to the 50-microseconds time window from the collision cell to the time-of-flight detector. For the convolution options, choose Tiernan's double integral, which includes the convolution over translational energy distributions between the ternary complex ion and the argon collision gas. For the numerical integration method, choose Gaussian Quadrature with pre-saved cross sections.
From the options for entering the molecular parameters, enter G to read the structural modeling file with the PM6 vibrational and rotational frequencies of the ternary complex. Reply Yes to the question:Is one of the reactants atomic? Write the location and name of the modeling file.
Enter 1 for the charge on ion and 1.664 for the polarizability of the argon gas. The mass of ion and mass of target are for the ternary complex and argon, respectively, and are automatically read from the GB5 text file. Enter 0 for harmonic vibrations.
Hit Enter to read the 1-D and 2-D rotational constants from the structural modeling file. Select default values of 0 for the hindered rotor treatments and 1 for molecule symmetry. Choose the default 300 Kelvin for reactant temperature.
Select Integration for the method for reduction of density of states array. Select Yes to truncate the energy distribution. Enter 40000 wave number for maximum energy for distribution, 2 wave number for bin size, and 32 for number of points in energy distribution.
For parameters for TCID/RRKM model, choose Yes for Change, enter 0 for Fixed Time, and 0.000050 seconds for the upper limit of the detection window. For product channel 1, select 1 for a single transition state from the dissociation channel options and 0 for none of the sequential dissociation. For the transition state type, choose 1 for orbiting.
Select G to read the modeling program files that contain the PM6 rotational and vibrational parameters for the amb-metal complex plus NTA products. Enter No for:Is one of the phase space limit transition state, or PSL TS, species atomic? Enter the location and name of the amb-metal complex file.
Use 1.062 for scale frequencies, hit Enter for the number of atoms, and enter No for:Is the molecule linear? Repeat the same for the modeling file that contains the vibrational and rotational frequencies of the NTA product. Enter the description of the orbiting TS.Enter 1.0 for charge of amb-metal ion, and then enter the polarizability and the dipole moment of NTA.
Select 0 Kelvin for rotational temperature and locked-dipole for the treatment of the orbiting transition state. Enter the average masses of the amb-metal ion and NTA. Hit Enter to read the 1-D and 2-D rotational constants from the modeling files.
Select 0 for hindered rotors, 1 for molecule symmetry, and 1 for reaction degeneracy. Enter the No Changes option. For product channel 2, select 1 for single transition state, 0 for none for the sequential dissociation, and 1 for orbiting for the transition state type.
Select G to read in modeling files that contain the PM6 rotational and vibrational parameters for the NTA-metal complex and amb products. Then, enter the description of the orbiting TS.Enter 1.0 for charge of NTA-metal complex ion, and enter the polarizability and the dipole moment of the amb. Select 0 Kelvin for the rotational temperature and locked-dipole for the treatment of the orbiting transition state.
Enter the average masses of the NTA-metal complex and amb products. Hit Enter to read the 1-D and 2-D rotational constants from the modeling files. Select 0 for hindered rotors, 1 for molecule symmetry, and 1 for reaction degeneracy.
Then, enter No Changes. To handle inactive 2-D rotations, select the default options for statistical angular momentum distribution and integrate P-E, G over J distribution. Use the default value of 32 in the number of points in the integration.
For the Model menu, select Optimize Parameters to Fit Data, and enter the minimum energy and maximum energy to begin and end the data fit, respectively. Select 1 for the weighting models'experimental standard deviations. Based on the data, select a minimum acceptable standard deviation of typically 0.01 to 0.001.
Use the default value for the E0 convergence limit and select No to hold any parameter at present value, 0.5 and 2.0 electronvolt for the lower and upper limits and 2 for the derivative evaluation method. From the Optimization menu, select Begin Optimization. The CRUNCH program will optimize the selected TCID model to the experimental data.
Finally, in the Model menu, select delta-H and S at T for the thermochemical evaluation of the two dissociation channels. The representative image shows the primary structures of alternative metal binding A and H peptides. The color highlights the potential metal binding sites.
The energy dependence for forming the amb-metal and NTA-metal product ions is shown here. The center-of-mass collision energy, where there is 50%dissociation of the amb-metal-NTA ternary complex, is included in the graphs. The representative images show the dynamics model for the energy-resolved TCID method.
The collisions between the ambH-zinc-NTA complex plus argon result in the dissociation to the ambH-zinc complex and free NTA, or NTA-zinc complex and free ambH products. The threshold energies E1 and E2 equate to the 0 Kelvin enthalpies of dissociation for the reactions ambH-zinc-NTA complex to ambH-zinc complex and free NTA, or ambH-zinc-NTA complex to NTA-zinc complex and free ambH, respectively. The PM6 geometry-optimized ternary amb-metal-NTA complexes of A and H are shown here.
These conformers were used in the TCID modeling of the experimental data and were selected from other candidate structures by comparing their PM6 electronic energies and how their calculated collision cross-sections compared to the IM-MS measured collision cross-sections. The energy-resolved TCID of the four amb-metal-NTA complexes is depicted in these images. For Species A and H, the product ions of amb-metal and NTA-metal with the convoluted CRUNCH threshold fits are shown here.
The energy values are the enthalpies of dissociation at 0 Kelvin for the reactions amb-metal-NTA to amb-metal and free NTA, or amb-metaL-NTA to NTA-metal and free amb. A comparison of the Gibbs free energies of association in kilojoules per mole and formation constants derived from the enthalpies of dissociation, net zero Kelvin, with statical mechanics calculations using the PM6 parameters are shown here. The reaction of NTA-nickel and free ambA to form the ambA-nickel-NTA complex exhibits the highest formation constant and represents the reaction where the ambA-tagged protein is immobilized by the NTA-nickel inside the affinity column.
CRUNCH fitting requires careful screening of reactants and products to obtain accurate threshold energies. Expertise in molecular modeling to obtain the structures and reliable molecular parameters is required. The overall methods described here could be developed for screening the effectiveness of molecules designed to bind metal cofactors and active sites of proteins to block the enzymatic functions.
