Name: ion mobility-mass spectrometry techniques for determining the structure and mechanisms of metal ion recognition and redox activity of metal binding oligopeptides
Uploaded: 2019-09-07T15:00:00
Description: Watch this Scientific Journal Video about Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopepti

This material is based upon work supported by the National Science Foundation under 1764436, NSF instrument support (MRI-0821247), Welch Foundation (T-0014), and computing resources from the Department of Energy (TX-W-20090427-0004-50)&#160;and L3 Communications. We thank the Bower&#8217;s group of University of California - Santa Barbara for sharing the Sigma program and Ayobami Ilesanmi&#160;for demonstrating the technique in the video.

1. Preparation of reagents
<ol>
	<li>Culture Methylosinus trichosporium OB3b, isolate the Cu(I)-free mb-OB3b18,22,23, freeze-dry the sample and store at -80 &#176;C until use.</li>
	<li>Synthesize the amb peptides (&#62;98% purity for amb1, amb2, amb4; &#62;70% purity for amb7), freeze-dry the samples, and store them at -80 &#176;C until use.</li>
	<li>Purchase &#62;98% puri...

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...

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 His2,3, (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(II) to ensure correct functioning.
Selective binding is often accomplished by acquisition and trafficking peptides, which control Zn(II) or Cu(I/II) ion concentrations4. Cu(I/II) is highly reactive and causes oxidative damage or adventitious binding to enzymes, so its free concentration is tightly regulated by copper chaperones and copper-regulating proteins that transport it safely to various locations in the cell and tightly control its homeostasis5,6. Disruption of copper metabolism or homeostasis is directly implicated in Menkes and Wilson&#8217;s disease7 as well as cancers7 and neural disorders, such as prion8 and Alzheimer&#8217;s disease9.
Wilson&#8217;s disease is associated with increased copper levels in the eyes, liver and sections of the brain, where the redox reactions of Cu(I/II) produces reactive oxygen species, causing hepatolenticular and neurological degeneration. Existing chelation therapies are the small thiol amino acid penicillamine and triethylenetetramine. Alternatively, the methanotrophic copper-acquisition peptides methanobactin (mb)10,11 exhibit therapeutic potential because of their high binding affinity for Cu(I)12. When the methanobactin (mb-OB3b) from Methylosinus trichosporium OB3b was studied in an animal model of Wilson&#8217;s disease, copper was efficiently removed from the liver and excreted through the bile13. In vitro experiments confirmed that mb-OB3b could chelate the copper from the copper metallothionein contained in the liver cytosol13. Laser ablation inductively coupled plasma mass spectrometry imaging techniques have investigated the spatial distribution of copper in Wilson&#8217;s disease liver samples14,15,16 and shown that mb-OB3b removes the copper with short treatment periods of only 8 days17.
The mb-OB3b will also bind with other metal ions, including Ag(I), Au(III), Pb(II), Mn(II), Co(II), Fe(II), Ni(II), and Zn(II)18,19. Competition for the physiological Cu(I) binding site is exhibited by Ag(I) because it can displace Cu(I) from the mb-OB3b complex, with both Ag(I) and Ni(II) also showing irreversible binding to Mb which cannot be displaced by Cu(I)19. Recently, a series of alternative methanobactin (amb) oligopeptides with the 2His-2Cys binding motif have been studied20,21, and their Zn(II) and Cu(I/II) binding properties characterized. Their primary amino acid sequences are similar, and they all contain the 2His-2Cys motif, Pro and an acetylated N-terminus. They mainly differ from mb-OB3b because the 2His-2Cys motif replaces the two enethiol oxazolone binding sites of mb-OB3b.
Electrospray ionization coupled with ion mobility-mass spectrometry (ESI-IM-MS) provides for a powerful instrumental technique for determining the metal-binding properties of peptides because it measures their mass-to-charge (m/z) and collision cross section (CCS) while conserving their mass, charge, and conformational shape from the solution-phase. The m/z and CCS relate to the peptides stoichiometry, protonation state, and conformational shape. Stoichiometry is determined because the identity and number of each element present in the species is explicitly identified. The overall charge of the peptide complex relates to the protonation state of the acidic and basic sites and the oxidation state of the metal ion(s). The CCS gives information of the conformational shape of the peptide complex because it measures the rotational averaged size which relates to the tertiary structure of the complex. The overall charge state of the complex is also a function of pH and affects the peptide&#8217;s metal ion binding affinity because the deprotonated basic or acidic sites such as the carboxyl, His, Cys and Tyr are also the potential binding sites for the metal ion. For the analyses, the peptide and metal ion are prepared in aqueous solutions with the pH adjusted by dilute aqueous acetic acid or ammonium hydroxide. This allows for the pH dependence and metal ion selectivity to be determined for the peptide. Furthermore, the m/z and CCS determined by ESI-IM-MS can be used with B3LYP/LanL2DZ molecular modeling to discover the type of metal ion coordination and tertiary structure of the complex. The results shown in this article reveal how ESI-IM-MS can characterize the selective chelating performance of a set of amb peptides and compare them to the copper-binding peptide mb-OB3b.

<table>
	<tbody>
		<tr>
			<td>acetonitrile HPLC-grade</td>
			<td>Fisher Scientific (www.Fishersci.com)</td>
			<td>A998SK-4</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>cobalt(II) chloride hexahydrate 99.99%</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>255599-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>copper(II) chloride 99.999%</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>203149-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>copper(II) nitrate hydrate 99.99%</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>229636-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>designed amb1,2,3,4,5,6,7 peptides</td>
			<td>Neo BioLab (neobiolab.com)</td>
			<td></td>
			<td>designed peptides were synthized by order</td>
		</tr>
		<tr>
			<td>designed amb5B,C,D,E,F peptides</td>
			<td>PepmicCo (www.pepmic.com)</td>
			<td></td>
			<td>designed peptides were synthized by order</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>Freeze-dried, purified, Cu(I)-free mb-OB3b</td>
			<td></td>
			<td></td>
			<td>cultured and isolated in the lab of Dr. DongWon Choi (Biology Department, Texas A&#38;M-Commerce)</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>Iron(III) Chloride Anhydrous 98%+</td>
			<td>Alfa Aesar (www.alfa.com)</td>
			<td>12357-09</td>
			<td></td>
		</tr>
		<tr>
			<td>lead(II) nitrate ACS grade</td>
			<td>Avantor (www.avantormaterials.com)</td>
			<td>128545-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>manganese(II) chloride tetrahydrate 99.99%</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>203734-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx 4.1</td>
			<td>Waters (www.waters.com)</td>
			<td></td>
			<td>software analysis program</td>
		</tr>
		<tr>
			<td>nickel chloride hexahydrate 99.99%</td>
			<td>Sigma-Aldrich (www.sigmaaldrich.com)</td>
			<td>203866-5G</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>silver nitrate 99.9%+</td>
			<td>Alfa Aesar (www.alfa.com)</td>
			<td>11414-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Synapt G1 HDMS</td>
			<td>Waters (www.waters.com)</td>
			<td></td>
			<td>quadrupole - ion mobility- time-of-flight mass spectrometer</td>
		</tr>
		<tr>
			<td>zinc chloride anhydrous</td>
			<td>Alfa Aesar (www.alfa.com)</td>
			<td>A16281</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Metal binding of amb1 
The IM-MS study20 of amb1 (Figure 1A) showed that both copper and zinc ions&#160;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<su...

Watch this Scientific Journal Video about Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopepti

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

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, ...

Research

Education

JoVE Journal

JoVE Core

Chemistry

Atoms and Elements

SCIENTISTS IN ACTION

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Analyzing the Photo-oxidation of 2-propanol at Indoor Air Level Concentrations Using Field Asymmetric Ion Mobility Spectrometry

We demonstrate a versatile protocol to be used for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) volatile ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

Surface Functionalization of Metal-Organic Frameworks for Improved Moisture Resistance

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Identification and Quantification of Decomposition Mechanisms in Lithium-Ion Batteries; Input to Heat Flow Simulation for Modeling Thermal Runaway

The risks and possible accidents related to the normal use of lithium-ion batteries remain a serious concern. In order to get a better understanding of ...