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

Summary

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.

Abstract

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’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’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.

Introduction

Copper and zinc ions are essential for living organisms and crucial to processes including oxidative protection, tissue growth, respiration, cholesterol, glucose metabolism, and genome reading¹. To enable these functions, groups such as the thiolate of Cys, imidazole of His^2,3, (more rarely) thioether of methionine, and carboxylate of Glu and Asp selectively incorporate metals as cofactors into the active sites of metalloenzymes. The similarity of these coordination groups raises an intriguing question regarding how the His and Cys ligands selectively incorporate either Cu(I/II) or Zn(I....

Protocol

1. Preparation of reagents

1. Culture Methylosinus trichosporium OB3b, isolate the Cu(I)-free mb-OB3b^18,22,23, freeze-dry the sample and store at -80 °C until use.
2. Synthesize the amb peptides (>98% purity for amb₁, amb₂, amb₄; >70% purity for amb₇), freeze-dry the samples, and store them at -80 °C until use.
3. Purchase >98% purity manganese(II) chloride, cobalt(II) chloride, nickle(II) chloride, copper(II) chloride, copper(II) nitrate, silver(I) nitrate, zinc(II) chloride, ir....

Results

Metal binding of amb₁
The IM-MS study²⁰ of amb₁ (Figure 1A) showed that both copper and zinc ions bound to amb₁ in a pH-dependent manner (Figure 2). However, copper and zinc bound to amb₁ through different reaction mechanisms at different coordination sites. For example, adding Cu(II) to amb₁ resulted in oxidation of amb₁ (amb

Discussion

Critical steps: conserving solution-phase behaviors for examination via ESI-IM-MS
Native ESI instrumental settings must be used that conserve the peptides stoichiometry, charge state, and conformational structure. For native conditions, the conditions in the ESI source such as the cone voltages, temperatures, and gas flows have to be optimized. Also, the pressures and voltages in the source, trap, ion mobility, and transfer traveling waves (especially the DC trap bias that controls injection voltag.......

Materials

Name	Company	Catalog Number	Comments
acetonitrile HPLC-grade	Fisher Scientific (www.Fishersci.com)	A998SK-4
ammonium hydroxide (trace metal grade)	Fisher Scientific (www.Fishersci.com)	A512-P500
cobalt(II) chloride hexahydrate 99.99%	Sigma-Aldrich (www.sigmaaldrich.com)	255599-5G
copper(II) chloride 99.999%	Sigma-Aldrich (www.sigmaaldrich.com)	203149-10G
copper(II) nitrate hydrate 99.99%	Sigma-Aldrich (www.sigmaaldrich.com)	229636-5G
designed amb1,2,3,4,5,6,7 peptides	Neo BioLab (neobiolab.com)		designed peptides were synthized by order
designed amb5B,C,D,E,F peptides	PepmicCo (www.pepmic.com)		designed peptides were synthized by order
Driftscope 2.1 software program	Waters (www.waters.com)		software analysis program
Freeze-dried, purified, Cu(I)-free mb-OB3b			cultured and isolated in the lab of Dr. DongWon Choi (Biology Department, Texas A&M-Commerce)
glacial acetic acid (Optima grade)	Fisher Scientific (www.Fishersci.com)	A465-250
Iron(III) Chloride Anhydrous 98%+	Alfa Aesar (www.alfa.com)	12357-09
lead(II) nitrate ACS grade	Avantor (www.avantormaterials.com)	128545-50G
manganese(II) chloride tetrahydrate 99.99%	Sigma-Aldrich (www.sigmaaldrich.com)	203734-5G
MassLynx 4.1	Waters (www.waters.com)		software analysis program
nickel chloride hexahydrate 99.99%	Sigma-Aldrich (www.sigmaaldrich.com)	203866-5G
poly-DL-alanine	Sigma-Aldrich (www.sigmaaldrich.com)	P9003-25MG
silver nitrate 99.9%+	Alfa Aesar (www.alfa.com)	11414-06
Waters Synapt G1 HDMS	Waters (www.waters.com)		quadrupole - ion mobility- time-of-flight mass spectrometer
zinc chloride anhydrous	Alfa Aesar (www.alfa.com)	A16281