This material is based upon work supported by the National Science Foundation under 1764436, NSF REU program (CHE-1659852), NSF instrument support (MRI-0821247), Physics and Astronomy Scholarship for Success (PASS) NSF project (1643567), Welch Foundation (T-0014), and computing resources from the Department of Energy (TX-W-20090427-0004-50) and L3 Communications. The authors thank Kent M. Ervin (University of Nevada - Reno) and Peter B. Armentrout (University of Utah) for sharing the CRUNCH program and for advice on fitting from PBA. The authors thank Michael T. Bower&#39;s group at the University of California - Santa Barbara for sharing the Sigma program.

NOTE: Figure 1 shows an overview of the protocol.
1. Preparation of reagents
<ol>
	<li>Purchase freeze-dried amb peptides (&#62;98% purity) and store them in a &#8722;80 &#176;C freezer.</li>
	<li>Purchase &#62;99% purity zinc(II) nitrate hexahydrate and nickel(II) nitrate hexahydrate. 
	CAUTION: Nickel(II) nitrate hexahydrate presents an environmental and health hazard.</li>
	<li>Purchase the nitrilotriacetic acid, poly-DL-alanine polymers...

Critical steps 
ES-IM-MS threshold collision-induced dissociation (TCID) analyses. The TCID used the transfer T-wave cell in the presence of argon as the collision cell. Prior to dissociation, the precursor ions are thermalized by low-energy collisions with nitrogen gas as they pass through the ion mobility (IM) cell. This results in a more reproducible energy-resolved TCID than is achieved by using the trap as the collision cell6,<sup class="xref"...

This study describes a new technique using a commercially available ion mobility-mass spectrometer that allows the determination of the relative thermochemistry for the dissociation of an alternative metal binding (amb) ternary metal complex [amb+M(II)+NTA], where M = Zn or Ni and NTA = nitrilotriacetic acid (Figure 1). These reactions model the dissociation of the amb-tagged recombinant protein attached to the NTA-immobilized metal during immobilized metal affinity chromatography (IMAC)1,2. As an example, this method is described using the amb heptapeptide tags of amb&#160;A and H (Figure 2) (chosen from the previous studies3,4,5,6,7,8,9,10,11,12) that exhibit Zn(II) and Ni(II)-binding properties and, thus, have potential applications as purification tags. However, the described process can be used to evaluate thermochemical energies in any organometallic system. These amb peptides have metal-binding sites in the Aa1-Aa2 and Aa6-Aa7 positions&#160;that compete with the carboxylate and amine sites of the NTA. The three central amb amino acids provide a spacer (Gly3), the hinge for the two arms (Pro4), and a long-distance &#960;-metal cation interaction (Tyr5).
The overall 1&#8722; charge state of the [amb+M(II)+NTA]- complexes is determined by the protonation state of their potential binding sites. Since there is Ni(II) or Zn(II) with the 2+ oxidation state, there must be a net of three deprotonated negatively-charged sites. The molecular modeling of the [amb+M(II)+NTA]- complexes predicts that these are two protons from the NTA and one proton from the amb (i.e., [amb-H+M(II)+NTA-2H]-). The product channels contain an ionic species and a neutral species (i.e., [NTA-3H+M(II)]- + amb or [amb-3H+M(II)]- + NTA). In the manuscript, &#34;-3H&#34; is excluded in the names of the complexes, but the reader should know that the -3H is implied. The instrument measures the relative intensities of the two ionic mass-to-charge (m/z) species. A major attribute of ES-IM-MS analyses is that it allows the examination of the reactivity of a specific m/z species, as utilized here and in previous amb studies3,4,5,6,7,8,9,10,11,12.
Acquiring thermochemical data for large complexes using collision-induced dissociation&#160;is a subject of significant interest13,14. Methodologies including the kinetic method are not conducive to fitting data over a range of energies, nor do they account for multi-collision environments15,16,17,18. Here, the threshold CID (TCID) method, developed using guided ion beam tandem mass spectrometry by Armentrout, Ervin, and Rodgers is applied19 to a new ES-IM-MS instrument platform utilizing traveling-wave ion guides. The TCID method allows for relative thermochemical analysis of the dissociation of the ternary complexes into their two product channels and includes a threshold law describing the transfer of collision energy between the translational energy of the reactant (ternary complex in this research) and an inert target gas (argon in this case). The method includes integration over the reactant&#39;s internal energy distribution20, the translational energy distributions between the reactant and target gas21, and the total angular momentum distributions22,23. A dissociation probability and statistical Rice-Ramsperger-Kassel-Marcus (RRKM) correction of the kinetic shifts resulting from the limited time window for observation of the products are included24. For two independent product channels, the competitive TCID method allows for the simultaneous fitting of the two competing product channels. Dissociation of the complex is through an orbiting transition state, which has the properties of the products but is held together by a locked-dipole25. The TCID method is incorporated into the CRUNCH program26, and the operation of the user interface is described here to evaluate the thermochemistry of the two dissociation channels of the ternary [amb+M(II)+NTA]- complexes. The CRUNCH program is available upon request from the developers26.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile HPLC-grade</td>
			<td>Fisher Scientific (www.Fishersci.com)</td>
			<td>A998SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Alternative metal binding (amb) peptides</td>
			<td>PepmicCo (www.pepmic.com)</td>
			<td></td>
			<td>designed peptides were synthized by order</td>
		</tr>
		<tr>
			<td>Ammonium acetate (ultrapure)</td>
			<td>VWR</td>
			<td>97061-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide (trace metal grade)</td>
			<td>Fisher Scientific (www.Fishersci.com)</td>
			<td>A512-P500</td>
			<td></td>
		</tr>
		<tr>
			<td>Driftscope 2.1 software program</td>
			<td>Waters (www.waters.com)</td>
			<td></td>
			<td>software analysis program</td>
		</tr>
		<tr>
			<td>Gaussian 09</td>
			<td>Gaussian</td>
			<td></td>
			<td>Electronic Structure Modeling Software</td>
		</tr>
		<tr>
			<td>GaussView</td>
			<td>Gaussian</td>
			<td></td>
			<td>Graphical Interface to Visualize Computations</td>
		</tr>
		<tr>
			<td>Glacial acetic acid (Optima grade)</td>
			<td>Fisher Scientific (www.Fishersci.com)</td>
			<td>A465-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Ion-scaled Lennard-Jones (LJ) method</td>
			<td>Sigma</td>
			<td></td>
			<td>Michael T. Bowers&#8217; group of University of California at Santa Barbara</td>
		</tr>
		<tr>
			<td>MassLynx 4.1</td>
			<td>Waters (www.waters.com)</td>
			<td></td>
			<td>software analysis program</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td>1.7 mL, polypropylene</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes</td>
			<td>VWR</td>
			<td>87003-298</td>
			<td>2.0 mL, polypropylene</td>
		</tr>
		<tr>
			<td>Ni(II) nitrate hexahydrate (99% purity)</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>A15540</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-DL-alanine</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>P9003-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Synapt G1 HDMS</td>
			<td>Waters (www.waters.com)</td>
			<td></td>
			<td>&#160;quadrupole - ion mobility- time-of-flight mass spectrometer</td>
		</tr>
		<tr>
			<td>Zn(II) nitrate hexahydrate (99%+ purity)</td>
			<td>Alfa Aesar (www.alfa.com)</td>
			<td>12313</td>
			<td></td>
		</tr>
	</tbody>
</table>

thermochemical studies of ni(ii) and zn(ii) ternary complexes using ion mobility-mass spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

The competitive collision-induced dissociation of the [amb+M(II)+NTA]- ternary complexes of A and H into [amb+M(II)]- + NTA or [NTA+M(II)]-&#160;+ amb,&#160;are shown in Figure 3. The amb is shown as either A or H and the M = Zn or Ni. The [A+Zn(II)+NTA]- ternary complex (Figure 3A) exhibits apparent thresholds of about 0.7 eV collision energy (CE) to ...

Watch this Scientific Journal Video about Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry at JoVE.com

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

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.

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

Abstract