Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry
Summary
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.
Abstract
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)]– + 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' 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 (ΔH0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (ΔH298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.
Introduction
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 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 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 π-metal cation interaction (Tyr5).
The overall 1− 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, "-3H" 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 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'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.
Protocol
Representative Results
Discussion
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"…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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's group at the University of California – Santa Barbara for sharing the Sigma program.
Materials
| Acetonitrile HPLC-grade | Fisher Scientific (www.Fishersci.com) | A998SK-4 | |
| Alternative metal binding (amb) peptides | PepmicCo (www.pepmic.com) | designed peptides were synthized by order | |
| Ammonium acetate (ultrapure) | VWR | 97061-014 | |
| Ammonium hydroxide (trace metal grade) | Fisher Scientific (www.Fishersci.com) | A512-P500 | |
| Driftscope 2.1 software program | Waters (www.waters.com) | software analysis program | |
| Gaussian 09 | Gaussian | Electronic Structure Modeling Software | |
| GaussView | Gaussian | Graphical Interface to Visualize Computations | |
| Glacial acetic acid (Optima grade) | Fisher Scientific (www.Fishersci.com) | A465-250 | |
| Ion-scaled Lennard-Jones (LJ) method | Sigma | Michael T. Bowers’ group of University of California at Santa Barbara | |
| MassLynx 4.1 | Waters (www.waters.com) | software analysis program | |
| Microcentrifuge Tubes | VWR | 87003-294 | 1.7 mL, polypropylene |
| Microcentrifuge Tubes | VWR | 87003-298 | 2.0 mL, polypropylene |
| Ni(II) nitrate hexahydrate (99% purity) | Sigma-Aldrich (www.sigmaaldrich.com) | A15540 | |
| Poly-DL-alanine | Sigma-Aldrich (www.sigmaaldrich.com) | P9003-25MG | |
| Waters Synapt G1 HDMS | Waters (www.waters.com) | quadrupole – ion mobility- time-of-flight mass spectrometer | |
| Zn(II) nitrate hexahydrate (99%+ purity) | Alfa Aesar (www.alfa.com) | 12313 |
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