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' Aa_1,2,6,7 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 

Protocol

NOTE: Figure 1 shows an overview of the protocol.

1. Preparation of reagents

1. Purchase freeze-dried amb peptides (>98% purity) and store them in a −80 °C freezer.
2. Purchase >99% purity zinc(II) nitrate hexahydrate and nickel(II) nitrate hexahydrate.
   CAUTION: Nickel(II) nitrate hexahydrate presents an environmental and health hazard.
3. Purchase the nitrilotriacetic acid, poly-DL-alanine polymers.......

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 cell^6,

Materials

Name	Company	Catalog Number	Comments
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