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.

The authors have no conflict of interest to disclose.

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 cell6,40. The thermalization of the [amb+M(II)+NTA]- prior to dissociation also allows the available internal energy of the ternary complex to be characterized using 298 K temperature. The dissociation in the transfer cell also means the ternary complex and its product ions have the same average arrival times at the detector, which was useful for identifying the dissociation of the ternary complex that only occurred in the transfer cell. Other regions where dissociation can occur are the ES source (sampling cone is kept at 25 V to avoid this) or at the entrance of the IM cell. The product ions produced by the dissociation of the ternary complex in these regions have different drift times from those produced in the transfer cell because the product ions are separated from the ternary complex in the IM cell. Those product ions were excluded from the analysis. In this protocol, only the integrated arrival time distributions for the precursor and product ions that are co-aligned are used to determine their intensities. The trap bias setting is the voltage that controls the injection voltage into the IM cell, which contributes to the CID at the entrance of the IM cell. The trap bias was set at 14 V, which kept the background dissociation to a minimum while not overly affecting the overall intensities. A previous study41 determined the effective temperature (upper limit) of the peptide dimer of leucine enkephalin to be 449 K at the entrance of the IM cell. However, the effective temperature decreased rapidly as the dimer passed down the IM cell. The arrival times of the amb complexes studied here exhibited Gaussian distributions, indicating they were thermalized as they passed down the IM cell.
ES-IM-MS collision cross sections (CCS) analyses. CCS drift times were found experimentally as the result of collisions with nitrogen. Those values were converted to helium-derived CCS drift times using a calibration curve of known standards. This is essential as the programs used to measure the CCS of the PM6 conformers require the more commonly used helium standards.
Modifications and troubleshooting of the technique 
CRUNCH input text file format. The input text file suitable for the CRUNCH program is shown in Figure 7. The headers in order from top to bottom are file location and version of CRUNCH; date; number of energies; number of data series excluding the first energies column; source file; mass of the precursor complex; mass of argon; temperature of experiment; date of creation; x-data designated as &#8722;1 (the center-of-mass collision energies); and the full width at half maximum (FWHM) of the ion beam. These values must be modified for each TCID experiment. The FWHM energy spread of the ion beam and energy zero should be determined by retarding potential analysis (RPA) by scanning the CE through low voltages and monitoring the total ion current. However, under the operating conditions of the IM in the current study, the ion current signal only decreased by about 50% when the transfer CE was set to its lowest value. The ion beam energy zero and FWHM could be measured only upon additional retardation by lowering the exit IM lens. In this latter case, the FWHM of the derivative of the RPA curve gave a typical ion energy spread of 1.5 V in the lab-frame or 0.035 eV in the center-of-mass frame13.
The pressures row relates to pressure inside the collision cell but is not used here. The pressures of argon in the collision cell can be varied and the TCID data can be measured at three pressures to extrapolate to single collision conditions. However, only one pressure was used in this study, and the pressure results in multiple collisions. Developing the new platform for a single collision is an area of ongoing research. Masses relate to the two product ions whose intensities are in the columns below. Dwells can be left as default. The five columns are the center-of-mass collision energies (designated &#8722;1); the mean of the ion intensities of the species with mass 898.30 u; the standard deviations of the ion intensities of species 898.30 u; the mean of the ion intensities of the species with mass 253.53 u; and the standard deviations of the ion intensities of species 253.53 u.
Molecular Modeling 
The number of conformers were narrowed initially by using models derived from previous studies9,10,11,12,13. CRUNCH fitting requires careful screening of reactants, activated molecules, and transition states to obtain accurate threshold energies. Previous research9,10,11,12,13 has included extensive screening of [amb+M(II)] conformers to obtain the structures with the parameters used in the CRUNCH modeling here. Only complexes with trans peptide bonds were used because only they are in agreement with IM-MS measured CCSHe10. The B3LYP and PM6 molecular modeling methods both predict the lowest energy [amb+M(II)]- conformer that exhibits Aa1-Cys2-Cys7 and carboxylate terminus coordination of Zn(II) or Ni(II)10,11,12,13. Familiarity with the behaviors of the known models allowed for the new conformers of [amb+M(II)+NTA]- to be determined more efficiently. To assist in conformer determination, as lower energy conformers were located by the PM6 method, they were filtered out and reassessed systematically until the most feasible, lowest energy conformers remained.
CRUNCH modeling 
Time-window for observing dissociation. In this study, the 50 &#956;s time window from the beginning of the transfer cell to the end of the TOF analyzer, where the multichannel plate detector is positioned, was used. It may be better to use the experimental time window between activation in the transfer cell and the entrance to the TOF mass analyzer because, if the activated ion dissociates during its time in the reflectron TOF, this metastable decay will be measured at a different m/z. However, in this study, the product ions observed in the mass spectra were all identifiable as the unmodified m/z species shown in Figure 3. This indicates that metastable decay was not an issue. Further research could investigate this by examining a known reaction with a high threshold and checking that the correct threshold energy is obtained using the 50 &#956;s time window and RRKM modelling.
Scaling factors for the vibrational frequencies. The NIST-recommended scaling factors for PM6 (1.062) vibrational frequencies were used. These were satisfactory for fitting the [A+Zn(II)+NTA]-, [A+Ni(II)+NTA]-, and [H+Zn(II)+NTA]- data. For some cases where the higher energy channel is entropically favored over the lower energy channel, it may be necessary to additionally scale the frequencies of the second channel. One approach is to scale the frequencies below 900 cm&#8722;1 (as these are the least accurate) to loosen the frequencies and make the TS more entropically favored.
Optimization of parameters. Using the Yes option to Hold any parameter at present value can be helpful to fit the data successfully.&#160;For the first fit, the E0(2) is held and the model TCID is fitted to the data by optimizing the CONST, E0(1), and N variables. Once a good fit is located, the parameters option and Hold any parameter at present value can be used to hold CONST, E0(1), and N, while allowing E0(2) to optimize to the data. Finally, once E0(2) is optimized, in the parameters option, all four parameters CONST, E0(1), E0(2), and N should be allowed to optimize to the data.
Energy range for fitting the selected TCID model to the experimental data. The energy range used to fit the experimental data should reproduce as much of the experimental intensity data as possible while maintaining a good fit in the threshold region. One can start by fitting the TCID model to a small energy range at the thresholds of the experimental data. One can choose a starting energy that exhibits the background intensity just prior to the rising intensity threshold behavior. Once the TCID fit is optimized to the experimental data range, the range should be increased by 0.1 eV and the fit should be optimized again. This procedure should be repeated to fit as much of the data range as possible while maintaining the fit of the threshold region.
Thermochemical Analyses. The thermochemical results from Delta H and S at T option should be compared with a series of different energy range fits to the data to estimate the standard deviation of the TCID model fit. Fits to compare should include smaller ranges that fit the initial rising threshold intensities well with those with greater ranges that include the higher energies as well.

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><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&amp;rsquo; 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>&amp;nbsp;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 produce [A+Zn(II)]- and about 0.9 eV to produce [NTA+Zn(II)]-. The dissociation of the [A+Ni(II)+NTA]- complex (Figure 3B) exhibits similar thresholds (~1.1 eV) for both the [NTA+Ni(II)]- and [A+Ni(II)]- products, with [NTA+Ni(II)]- increasing to 90% relative intensity, while the intensities of [A+Ni(II)]-&#160;do not rise above 18%. For the [H+Zn(II)+NTA]- ternary complex (Figure 3C), the main product is [H+Zn(II)]- rising from a threshold of about 0.6 eV to about 85% relative intensity, and at energies above 1.0 eV, the [NTA+Zn(II)]-&#160;rises to about 30%. There is also a channel for water loss from [H-H2O+Zn(II)]-. For [H+Ni(II)+NTA]- (Figure 3D), the [H+Ni(II)]- rises from a threshold of about 0.9 eV to about 40% relative intensity, while the [NTA+Ni(II)]- rises from ~1.0 eV to about 80%. Included on the graphs is the CE where the ternary complex is 50% dissociated. The Ni(II) ternary complexes require 0.31-0.37 eV higher CE than their Zn(II) ternary complex counterparts to be 50% dissociated. This suggests the Ni(II) complexes are more stable and require higher CE to dissociate, which is further investigated using the TCID technique.
Figure 4 illustrates the competitive TCID method, which allows for the simultaneous fitting of the two competing product channels.
[amb+M(II)+NTA] &#8594; [amb+M(II)]- + NTA (1)
[amb+M(II)+NTA] &#8594; [NTA+M(II)]- + amb (2)
The potential energy surface (PES) illustrates the energized ternary complex dissociating into the competing product channels and shows the PM6 geometry-optimized species used to model the dissociation of [ambH+Zn(II)+NTA]-. Included in the PES are the density of states of the ternary complex and the sum of states of the products. The 0 K threshold energies, E1 and E2, equate to the 0 K enthalpy change for reactions 1 and 2.
Figure 5 shows the structures of the other three geometry-optimized ternary complexes used in this study. These species were chosen based on their predicted electronic and zero-point energies and their agreement with the IM-MS-measured collision cross-sections (CCSHe). Table 1 shows there is an agreement between the ternary complexes LJ CCSHe and the experimental IM-MS CCSHe because they agree within their mutual uncertainties. The conformations of the [amb+M(II)]&#160;and amb were based on the findings of our previous DFT modeling3,4,5,6. The molecular parameters of these PM6 conformers were used in the TCID modeling of the energy-resolved dissociations of the ternary complexes, including their ro-vibrational frequencies for calculating their density and sum of states.
Figure 6 shows the convoluted CRUNCH TCID threshold fits to the energy-resolved product intensities. The convoluted fits include the available energy and angular momentum distributions of the [amb+M(II)+NTA]- + Ar reactants. The unconvoluted fits (not shown) predicted the 0 K change in enthalpies (&#916;H0) for the dissociation of the ternary complex, and Table 2 shows the &#916;H0 and &#916;H298 (kJ/mol) for reactions 1 and 2. For the dissociation of the Zn(II) ternary complexes, both A and H exhibit &#916;H0 for reaction 1,&#160;which are 31 kJ/mol and 15 kJ/mol lower than the &#916;H0 for reaction 2, respectively, indicating both A and H have greater Zn(II) affinity than the NTA. The [A+Ni(II)+NTA]-&#160;ternary complex exhibits &#916;H0 = 146 and 148 kJ/mol for reactions 1 and 2, respectively, indicating A and NTA have similar affinities for Ni(II). However, the dissociation of [H+Ni(II)+NTA]-&#160;shows&#160;the &#916;H0 for reaction 1&#160;is 36 kJ/mol lower than for reaction 2, indicating H has a greater Ni(II) affinity than the NTA. Overall, the [amb+Ni(II)+NTA]- complexes exhibit higher dissociation enthalpies than their [amb+Zn(II)+NTA]-&#160;counterparts, with the exception of A dissociating into [NTA+Ni(II)]-. Table 3 shows the Gibbs free energies (&#916;G298) of association and the formation constants (K) for the reverse reactions:
[amb+M(II)]- + NTA &#8594; [amb+M(II)+NTA]- (3)
[NTA+M(II)]- + amb &#8594; [amb+M(II)+NTA]- (4)
Table 3 demonstrates that the formation of the Ni(II) ternary complexes is more exergonic and exhibits larger formation constants K than the Zn(II) complexes in all cases. Reaction 4 (i.e., the amb tag association with the NTA metal ion complex) is of particular interest as it represents the amb-tagged recombinant protein binding to the NTA-immobilized metal ion inside the IMAC column. Reaction 4 for the formation of [ambA+Ni(II)+NTA] exhibits the most spontaneous &#916;G298 = 53.1 kJ/mol and the highest formation constant, K = 2.01 x 109.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63722/63722fig01.jpg" /> 
Figure 1: Overview of the ES-IM-MS TCID method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63722/63722fig02.jpg" /> 
Figure 2: The primary structures of amb&#160;A and H peptides. Color highlights the potential metal-binding sites. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63722/63722fig03.jpg" /> 
Figure 3: The center-of-mass, energy-resolved (eV) threshold collision-induced dissociation of [amb+M(II)+NTA]-. The energy-dependence of the product ions [amb+M(II)]- [NTA+M(II)]- and [amb-H2O+Zn(II)]- is shown. The center-of-mass collision energy, where there is 50% dissociation of the [amb+M(II)+NTA]- ternary complex, is included on the graphs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63722/63722fig04.jpg" /> 
Figure 4: The model for the energy-resolved TCID method. The collisions between [ambH+Zn(II)+NTA]- + argon result in the dissociation to the [ambH+Zn(II)]-&#160;+ NTA or [NTA+Zn(II)]- + ambH products. The threshold energies E1 and E2 equate to the 0 K enthalpies of dissociation (&#916;H0) for the reactions [ambH+Zn(II)+NTA]- &#8594; [ambH+Zn(II)]-&#160;+ NTA or [ambH+Zn(II)+NTA]-&#160;&#8594; [NTA+Zn(II)]-&#160;+ ambH, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63722/63722fig05.jpg" /> 
Figure 5: The PM6 geometry-optimized ternary [amb+M(II)+NTA]- complexes of A and H. Conformers used in the TCID modeling of the experimental data. These conformers were selected from other candidate structures by comparing their PM6 electronic energies and how their LJ collision cross-sections (CCSHe) compared to the IM-MS measured CCSHe. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63722/63722fig06v2.jpg" /> 
Figure 6: The energy-resolved, collision-induced dissociation of [amb+M(II)+NTA]-. For species A and H, the product ions of [amb+M(II)]- and [NTA+M(II)]- with the convoluted CRUNCH threshold fits are shown. The energy (eV) values shown are the enthalpies of dissociation at 0 K for the reactions [amb+M(II)+NTA]-&#160;&#8594; [amb+M(II)]- + NTA or [amb+M(II)+NTA]- &#8594; [NTA+M(II)]- + amb. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig06v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63722/63722fig07.jpg" /> 
Figure 7: The format for the CRUNCH text input file. The file contains the mean relative intensities and their standard deviations of the product ions formed as a function of center-of-mass collision energy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/63722fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>amb</td>
			<td colspan="2">[amb+Zn(II)+NTA]-</td>
			<td colspan="2">[amb+Ni(II)+NTA]-</td>
		</tr>
		<tr>
			<td></td>
			<td>PM6</td>
			<td>Exp.a</td>
			<td>PM6</td>
			<td>Exp.a</td>
		</tr>
		<tr>
			<td>A</td>
			<td>214&#177;2</td>
			<td>214</td>
			<td>219&#177;2</td>
			<td>218</td>
		</tr>
		<tr>
			<td>H</td>
			<td>211&#177;5</td>
			<td>216</td>
			<td>212&#177;3</td>
			<td>215</td>
		</tr>
		<tr>
			<td colspan="5">a ES-IM-MS CCSHe measurements have uncertainties of &#177;4 &#197;2.</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Comparison of LJ collision cross-sections of the PM6 conformers of [amb+M(II)+NTA]-. Theoretical cross-sections of the PM6 conformers are compared with the experimental CCSHe measured with ES-IM-MS.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">[amb+Zn(II)+NTA]-&#160;&#8594;</td>
			<td colspan="4">[amb+Ni(II)+NTA]-&#160;&#8594;</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">[amb+Zn(II)]-&#160;+ NTA</td>
			<td colspan="2">[NTA+Zn(II)]-&#160;+ amb</td>
			<td colspan="2">[amb+Ni(II)]-&#160;+ NTA</td>
			<td colspan="2">[NTA+Ni(II)]-&#160;+ amb</td>
		</tr>
		<tr>
			<td>amb</td>
			<td>&#916;H0</td>
			<td>&#916;H298</td>
			<td>&#916;H0</td>
			<td>&#916;H298</td>
			<td>&#916;H0</td>
			<td>&#916;H298</td>
			<td>&#916;H0</td>
			<td>&#916;H298</td>
		</tr>
		<tr>
			<td>A</td>
			<td>118</td>
			<td>127</td>
			<td>149</td>
			<td>182</td>
			<td>146</td>
			<td>171</td>
			<td>148</td>
			<td>154</td>
		</tr>
		<tr>
			<td>H</td>
			<td>96.4</td>
			<td>92.3</td>
			<td>111</td>
			<td>115</td>
			<td>125</td>
			<td>140</td>
			<td>161</td>
			<td>216 
			&#160;</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;Thermochemical results from the TCID analyses. The energy-dependent reactions [amb+M(II)+NTA]- &#8594; [amb+M(II)]- + NTA or [amb+M(II)+NTA]- &#8594; [NTA+M(II)]- + amb, showing the 0 K enthalpies of dissociation (&#916;H0) derived from the unconvoluted TCID model fit, and 298 K enthalpies of dissociation (&#916;H298) derived from &#916;H0 and statistical mechanics thermal corrections using the PM6 rotational and vibrational frequencies. Values are given in kJ/mol.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">[amb+Zn(II)]-&#160;&#160;+ NTA &#8594;</td>
			<td colspan="2">[NTA+Zn(II)]-&#160;+ amb &#8594;</td>
			<td colspan="2">[amb+Ni(II)]-&#160;+ NTA &#8594;</td>
			<td colspan="2">[NTA+Ni(II)]-&#160;+ amb &#8594;</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">[amb+Zn(II)+NTA]-</td>
			<td colspan="2">[amb+Zn(II)+NTA]-</td>
			<td colspan="2">[amb+Ni(II)+NTA]-</td>
			<td colspan="2">[amb+Ni(II)+NTA]-</td>
		</tr>
		<tr>
			<td>amb</td>
			<td>&#916;G298</td>
			<td>K</td>
			<td>&#916;G298</td>
			<td>K</td>
			<td>&#916;G298</td>
			<td>K</td>
			<td>&#916;G298</td>
			<td>K</td>
		</tr>
		<tr>
			<td>A</td>
			<td>-34.0</td>
			<td>9.05 x 105</td>
			<td>-21.8</td>
			<td>6.59 x 103</td>
			<td>-45.7</td>
			<td>1.01 x 108</td>
			<td>-53.1</td>
			<td>2.01 x 109</td>
		</tr>
		<tr>
			<td>H</td>
			<td>-29.3</td>
			<td>1.36 x 105</td>
			<td>-30.2</td>
			<td>&#160;1.95 x 105</td>
			<td>-47.0</td>
			<td>1.71 x 108</td>
			<td>-31.1</td>
			<td>2.81 x 105 
			&#160;</td>
		</tr>
	</tbody>
</table>
Table 3: Gibbs free energies of association (&#916;G298) and equilibrium formation constants (K).&#160;&#916;G298 and K&#160;at 298 K for the reverse reactions [amb+M(II)]-&#160;+ NTA &#8594; [amb+M(II)+NTA]- and [NTA+M(II)]- + amb &#8594; [amb+M(II)+NTA]-. Derived from &#916;H298 and statistical mechanics entropy calculations using the PM6 rotational and vibrational frequencies. Values for &#916;G298 are in kJ/mol.
Supplementary File. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63722/Supplementary-Protocol-Gaussian.docx" target="_blank">Please click here to download this File.</a>

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 ...

thermochemical-studies-ni-ii-zn-ii-ternary-complexes-using-ion

Nanyang Technological University

Research

JoVE Journal

Chemistry

2.2K Views.  Texas A&M University-Commerce. Deze nieuwe techniek combineert ionenmobiliteit-massaspectrometrie met moleculaire modellering en reactiedynamicatheorie om de relatieve thermochemie van twee concurrerende dissociatiereacties van een ternair complex te bepalen. De combinatie van deze technieken karakteriseert de reactieroutes van reactanten en producten, conformatiestructuren en de affiniteit van het ligand om een ternair complex met het metaalion te vormen. Dit onderzoek modelleert de reactiviteit van twee potentiële pepti...

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

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of molecular iodine to samarium metal in THF.1,2-3 It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2 can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12 One of the fascinating features of SmI-2-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14 Additives commonly utilized to fine tune the reactivity of SmI2 can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2, FeCl3, etc).3
Understanding the mechanism of SmI2 reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15 Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12 and have been utilized in key steps of the synthesis of large natural products.16,17 Previous studies on the effect of additives on SmI2 reactions have shown that HMPA enhances the reduction potential of SmI2 by coordinating to the samarium metal center, producing a more powerful,13-14,18 sterically encumbered reductant19-21 and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22 In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27 Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2 reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2 reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2-initiated reactions is described.

Preparation and Use of Samarium Diiodide SmI2 in Organic Synthesis: The Mechanistic Role of HMPA and NiII Salts in the Samarium Barbier Reaction

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of ...

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

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 of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

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, ...

Elemental-sensitive Detection of the Chemistry in Batteries through Soft X-ray Absorption Spectroscopy and Resonant Inelastic X-ray Scattering

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid based on volatile solar and wind sources. The pressing demand of developing high-performance electrochemical energy storage solutions, i.e., batteries, relies on both fundamental understanding and practical developments from both the academy and industry. The formidable challenge of developing successful battery technology stems from the different requirements for different energy-storage applications. Energy density, power, stability, safety, and cost parameters all have to be balanced in batteries to meet the requirements of different applications. Therefore, multiple battery technologies based on different materials and mechanisms need to be developed and optimized. Incisive tools that could directly probe the chemical reactions in various battery materials are becoming critical to advance the field beyond its conventional trial-and-error approach. Here, we present detailed protocols for soft X-ray absorption spectroscopy (sXAS), soft X-ray emission spectroscopy (sXES), and resonant inelastic X-ray scattering (RIXS) experiments, which are inherently elemental-sensitive probes of the transition-metal 3d and anion 2p states in battery compounds. We provide the details on the experimental techniques and demonstrations revealing the key chemical states in battery materials through these soft X-ray spectroscopy techniques.

Here, we present a protocol for typical experiments of soft X-ray absorption spectroscopy (sXAS) and resonant inelastic X-ray scattering (RIXS) with applications in battery material studies.

Energy storage has become more and more a limiting factor of today's sustainable energy applications, including electric vehicles and green electric grid ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Biology

Molecular Orbital (MO) Theory

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes featuring the ligand 1,1&#39;-bis(diphenylphosphino)ferrocene (dppf): M(dppf)Cl2, where M = Ni or Pd. While both of these transition metal complexes are 4-coordinate, they exhibit different geometries at the metal center. Using molecular orbital (MO) theory in conjunction with 1H NMR and Evans method, we will determine the geometry of these two compounds.

Molecular Orbital MO Theory: Predicting Geometry of Transition Metal Complexes

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes ...

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

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