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% purity manganese(II) chloride, cobalt(II) chloride, nickle(II) chloride, copper(II) chloride, copper(II) nitrate, silver(I) nitrate, zinc(II) chloride, iron(III) chloride and lead(II) chloride.</li>
	<li>Purchase the poly-DL-alanine polymers used as calibrants for measuring the collision cross sections of the amb species and HPLC grade or higher ammonium hydroxide, glacial acetic acid, and acetonitrile.</li>
</ol>
2. Preparation of stock solution
<ol>
	<li>Peptide stock solution
	<ol>
		<li>Weigh accurately, using at least three significant figures, the mass of 10.0&#8211;20.0 mg of the mb-OB3b or amb in a 1.7 mL plastic vial. 
		NOTE: The weighed mass should yield either 12.5 mM or 1.25 mM, depending on the solubility of the peptide, when 1.00 mL of deionized (DI) water is added.</li>
		<li>Using a pipet, add 1.00 mL of deionized water (&#62;17.8 M&#937; cm) to the weighed peptide sample to yield either the 12.5 mM or 1.25 mM solution. Place cap securely and mix thoroughly with at least 20 inversions.</li>
		<li>Using a micropipet dispense 50.0 &#956;L aliquots from the peptide sample into individually labelled 1.5 mL vials and store them at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Metal ion stock solutions
	<ol>
		<li>Weigh accurately, using at least three significant figures, the mass of 10.0&#8211;30.0 mg of the metal chloride or silver nitrate in a 1.7 mL vial. 
		NOTE: The weighed mass should yield 125 mM when 1.00 mL of DI water is added.</li>
		<li>Add the 1.00 mL of DI water to the weighed metal sample in the 1.7 mL vial to yield the 125 mM solution. Place cap securely and mix thoroughly with at least 20 inversions.</li>
	</ol>
	</li>
	<li>Ammonium hydroxide stock solutions: prepare a 1.0 M acetic acid solution by diluting 57 &#956;L of the 99.5% acetic acid solution with DI water to a final volume of 1.00 mL. Prepare a 1.0 M ammonium hydroxide solution by diluting 90 &#956;L of the 21% ammonium hydroxide solution with DI water to a final volume of 1.00 mL. Make two successive dilutions of each solution by taking 100 &#956;L of the 1.0 M solutions to prepare 0.10 M and 0.010 M acetic acid and ammonium hydroxide solutions.</li>
	<li>Poly-DL-alanine stock solution: prepare the poly-DL-alanine (PA) by weighing 1.0 mg of PA and dissolving in 1.0 mL of DI water to give 1,000 ppm. Mix thoroughly. Using a micropipet, dispense 50.0 &#956;L aliquots, and place each into a 1.7 mL vial and store at -80 &#176;C.</li>
</ol>
3. Electrospray-ion mobility-mass spectrometry analysis
<ol>
	<li>Clean the ESI entrance tubing and needle capillary thoroughly with about 500 &#956;L of 0.1 M glacial acetic acid, 0.1 M ammonium hydroxide, and finally DI water.</li>
	<li>Thaw a 50.0 &#956;L aliquot of the 1,000 ppm PA stock solution and dilute it with 450 &#956;L of DI water to give a 100 ppm PA. Pipet 100.0 &#956;L of this solution and dilute it to 1.00 mL with 500 &#956;L of DI water and 500 &#956;L of acetonitrile to give 10 ppm PA solution.</li>
	<li>Collect the negative and positive ion IM-MS spectra of the 10 ppm PA solution for 10 min each using native ESI-IM-MS conditions as described in the discussion section.</li>
	<li>Thaw a 50.0 &#956;L aliquot of the 12.5 mM or 1.25 mM amb stock solution and make successive dilutions with DI water to give a final concentration of 0.125 mM amb. Mix thoroughly each dilution.</li>
	<li>Pipet 100.0 &#956;L of the 125 mM metal ion stock solution, place in a 1.7 mL vial and dilute to 1.00 mL with DI water to give 12.5 mM metal ion. Repeat with two more successive dilutions to give a final 0.125 mM metal ion concentration. Mix thoroughly each dilution.</li>
	<li>Pipet 200.0 &#956;L of the 0.125 mM amb into a 1.7 mL vial, dilute with 500 &#956;L of DI water, and mix the solution thoroughly.</li>
	<li>Adjust the pH of the sample to 3.0 by adding 50 &#956;L of 1.0 M acetic acid solution.</li>
	<li>Add 200.0 &#956;L of the 0.125 mM metal ion to the pH-adjusted sample. Add DI water to yield a final volume of 1.00 mL of the sample, mix thoroughly, and allow the sample to equilibrate for 10 min at RT.</li>
	<li>Using a blunt nose syringe take 500 &#956;L of the sample and collect the negative and positive ion ES-IM-MS spectra for 5 min each. Use the remaining 500 &#956;L of the sample to record its final pH using a calibrated micro pH electrode.</li>
	<li>Repeat steps 3.6&#8211;3.9, while modifying step 3.7 to adjust the pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 by adding new volumes of the 0.010 M, 0.10 M, or 1.0 M acetic acid or ammonium hydroxide solutions.</li>
	<li>Collect the negative and positive ion ESI-IM-MS spectra of the 10 ppm PA solution for 10 min each.</li>
</ol>
4. Preparation of the metal ion titration of amb samples
<ol>
	<li>Follow the steps described in steps 3.1&#8211;3.5.</li>
	<li>Pipet 200.0 &#956;L of the 0.125 mM amb into a 1.7 mL vial, dilute with 500.0 &#956;L of DI water and mix the solution thoroughly.</li>
	<li>Adjust the pH of the sample to pH = 9.0 by adding 80 &#956;L of the 0.010 M ammonium hydroxide solution.</li>
	<li>Add 28 &#956;L of the 0.125 mM metal ion solution to give 0.14 molar equivalents of the metal ion, add DI water to make the final volume of the sample 1.00 mL, mix thoroughly, and allow the sample to equilibrate for 10 min at RT.</li>
	<li>Using a blunt nose syringe take 500 &#956;L of the sample and collect the negative and positive ion ESI-IM-MS spectra for 5 min each. Use the remaining 500 &#956;L of the sample to record its final pH using a calibrated micro pH electrode.</li>
	<li>Repeat steps 4.2&#8211;4.5, while modifying step 4.3 to add an appropriate volume of the 0.125 mM metal ion solution to give either 0.28, 0.42, 0.56, 0.70, 0.84, 0.98, 1.12, 1.26, or 1.40 molar equivalents.</li>
	<li>Collect the negative and positive ion IM-MS spectra of the 10 ppm PA solution for 10 min each.</li>
</ol>
5. Analysis of ESI-IM-MS pH titration data
<ol>
	<li>From the IM-MS spectra identify which charged species of ambs are present by matching them to their theoretical m/z isotope patterns.
	<ol>
		<li>Open MassLynx and click on Chromatogram to open the Chromatogram window.</li>
		<li>Go to the File menu and Open to locate and open the IM-MS data file.</li>
		<li>Extract the IM-MS spectrum by right-clicking and dragging across the chromatogram and releasing. The spectrum window will open showing the IM-MS spectrum.</li>
		<li>In the spectrum window, click on Tools and Isotope model. In the isotope modeling window, enter the molecular formula of the amb species, check the Show charged ion box, and enter the charge state. Click OK.</li>
		<li>Repeat to identify all the species in the IM-MS spectrum and record their m/z isotope range.</li>
	</ol>
	</li>
	<li>For each amb species, separate any coincidental m/z species and extract their arrival time distributions (ATD) using their m/z isotope patterns to identify them.
	<ol>
		<li>In MassLynx click on DriftScope to open the program. In DriftScope click on File and Open to locate and open the IM-MS data file.</li>
		<li>Use the mouse and left-click to zoom in on m/z isotope pattern of the amb species.</li>
		<li>Use the Selection tool and left mouse button to select the isotope pattern. Click the Accept current selection button.</li>
		<li>To separate any coincidental m/z species use the Selection tool and left mouse button to select the ATD time-aligned with isotope pattern of the amb species. Click the Accept current selection button.</li>
		<li>To export the ATD, go to File | Export to MassLynx, then select Retain Drift Time and save file in appropriate folder.</li>
	</ol>
	</li>
	<li>Determine the centroid of the ATD and integrate the area under the ATD curve as a measure of the species population.
	<ol>
		<li>In the Chromatogram window of MassLynx open the saved exported file. Click on Process | Integrate from the menu. Check the ApexTrack Peak Integration box and click OK.</li>
		<li>Record the centroid ATD (tA) and the integrated area as shown on the Chromatogram window. Repeat for all saved amb and PA IM-MS data files.</li>
	</ol>
	</li>
	<li>Use the integrated ATD for all extracted amb species of either the positive or negative ions at each titration point to normalize to a relative percentage scale.
	<ol>
		<li>Enter the identities of the amb species and their integrated ATD at each pH into a spreadsheet.</li>
		<li>For each pH, use the sum of the integrated ATDs to normalize the individual amb&#8217;s ATD to a percentage scale.</li>
		<li>Plot the percent intensities of each amb species vs. pH in a graph to show how the population of each species varies as a function of pH.</li>
	</ol>
	</li>
</ol>
6. Collision cross-sections
<ol>
	<li>Using a spreadsheet, convert the CCSs (&#937;) of PA negative25,26 and positive27 ions measured in He buffer gas28 to corrected CCS (&#937;c) using Equation 1 below, where: z = ion charge; ec = electron charge (1.602&#215;10-19 C); mN2 = mass of N2 gas (Da); and mion = ion mass.29</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/60102/60102equ01.jpg" />
<ol start="2">
	<li>Convert the average arrival times (tA) of the PA calibrants and amb species into drift times (tD) using Equation 2 below, where: c = the enhanced duty cycle delay coefficient (1.41), and m/z is the mass-to-charge of the peptide ion.</li>
</ol>
<img alt="Equation 2" src="/files/ftp_upload/60102/60102equ02.jpg" />
<ol start="3">
	<li>Plot the PA calibrants&#8217; tD vs. their &#937;c. Then, using a least-squares regression fit of Equation 3 shown below, determine the A&#39; and B values, where: A&#39; is the correction for the temperature, pressure, and electric field parameters; and B compensates for the nonlinear effect of the IM device.</li>
</ol>
<img alt="Equation 3" src="/files/ftp_upload/60102/60102equ03.jpg" />
<ol start="4">
	<li>Using these A&#39; and B values and the centroid tD value from the ATD of the ambs determine their &#937;c using Equation 3 and their &#937; using Equation 1. This method provides CCSs for the peptide species with estimated absolute errors of about 2%25,26,27.</li>
</ol>
7. Computational methods
<ol>
	<li>Use the B3LYP/LanL2DZ level of theory, comprising of the Becke 3-parameter hybrid functionals30 and the Dunning basis set31 and electron core potentials32,33,34 to locate geometry-optimized conformers for all possible types of coordinations of the observed m/z amb species35. 
	NOTE: For details of how to build and submit calculations refer to the GaussView usage in Supplementary File.</li>
	<li>Compare the predicted free energy of each of the conformers and calculate their theoretical CCSs using the ion-scaled Lennard-Jones (LJ) method from the Sigma program36.</li>
	<li>From the lowest free energy conformers determine which conformer exhibits the LJ CCS which agrees with the IM-MS measured CCS to identify the tertiary structure and type of coordination for the conformers observed in the experiment.</li>
</ol>

The authors have nothing to disclose.

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, ...

ion-mobility-mass-spectrometry-techniques-for-determining-structure

Research

Education

JoVE Journal

JoVE Core

Chemistry

Atoms and Elements

SCIENTISTS IN ACTION

9.1K Views.  Texas A&M University-Commerce. 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.

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

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 reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

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

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

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; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line 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 organic carbons (VOCs), illustrating this with a titanium dioxide based catalyst, and the VOC 2-propanol. The protocol takes advantage of field asymmetric ion mobility spectroscopy (FAIMS), an analysis tool that is capable of continuously identifying and monitoring the concentration of VOCs such as 2-propanol and acetone at the ppb level. The continuous nature of FAIMS allows detailed kinetic analysis, and long-term reactions, offering a significant advantage over gas chromatography, a batch process traditionally used in air purification characterization. The use of FAIMS in photocatalytic air purification has only recently been used for the first time, and with the protocol illustrated here, the flexibility in allowing alternative VOCs and photocatalysts to be tested using comparable protocols offers a unique system to elucidate photocatalytic air purification reactions at low concentrations.

A protocol for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) model volatile organic carbons such as 2-propanol is described.

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, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

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

Simple Methods for the Preparation of Non-noble Metal Bulk-electrodes for Electrocatalytic Applications

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and composition of the material was characterized via powder X-ray diffraction (PXRD), M&#246;ssbauer spectroscopy (MB), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and energy dispersive X-ray spectroscopy (EDX). Two preparation methods of pentlandite bulk electrodes are presented. In the first approach a piece of synthetic pentlandite rock is directly contacted via a wire ferrule. The second approach utilizes pentlandite pellets, pressed from finely ground powder, which is immobilized in a Teflon casing. Both electrodes, whilst being prepared by an additive-free method, reveal high durability during electrocatalytic conversions in comparison to common drop-coating methods. We herein showcase the striking performance of such electrodes to accomplish the hydrogen evolution reaction (HER) and present a standardized method to evaluate the electrocatalytic performance by electrochemical and gas chromatographic methods. Furthermore, we report stability tests via potentiostatic methods at an overpotential of 0.6 V to explore the material limitations of the electrodes during electrolysis under industrial relevant conditions.

A facile preparation method of electrodes using the bulk material Fe4.5Ni4.5S8 is presented. This method provides an alternative technique to conventional electrode fabrication and describes prerequisites for unconventional electrode materials including a straightforward electrocatalytic testing method.

The rock material pentlandite with the composition Fe4.5Ni4.5S8 was synthesized via high temperature synthesis from the elements. The structure and ...

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 of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

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. However, the main issue limiting their applicability is their poor stability in humid conditions. The common methods to overcome this problem involve the formation of strong metal-linker bonds by using highly charged metals, which is limited to a number of structures, the introduction of alkylic groups to the framework by post-synthetic modification (PSM) or chemical vapour deposition (CVD) to enhance overall hydrophobicity of the framework. These last two usually provoke a drastic reduction of the porosity of the material. These strategies do not permit to exploit the properties of the MOF already available and it is imperative to find new methods to enhance the stability of MOFs in water while keeping their properties intact. Herein, we report a novel method to enhance the water stability of MOF crystals featuring Cu2(O2C)4&#160;paddle-wheel units, such as HKUST (where HKUST stands for Hong Kong University of Science &#38; Technology), with the catechols functionalized with alkyl and fluoro-alkyl chains. By taking advantage of the unsaturated metal sites and the catalytic catecholase-like activity of CuII ions, we are able to create robust hydrophobic coatings through the oxidation and subsequent polymerization of the catechol units on the surface of the crystals under anaerobic and water-free conditions without disrupting the underlying structure of the framework. This approach not only affords the material with improved water stability but also provides control over the function of the protective coating, which enables the development of functional coatings for the adsorption and separations of volatile organic compounds. We are confident that this approach could also be extended to other unstable MOFs featuring open metal sites.

Robust functional catechol coatings were produced in one step by direct reaction of the material known as HKUST with synthetic catechols under anaerobic conditions. The formation of homogeneous coatings surrounding the entire crystal is ascribed to the biomimetic catalytic activity of Cu(II) dimers on the external surface of the crystals.

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 spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.

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 many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

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 thermal runaway (TR), the exothermic decomposition reactions in anode and cathode were studied, using a Simultaneous Thermal Analysis (STA)/Gas Chromatography-Mass Spectrometry (GC-MS)/Fourier Transform Infrared (FTIR) spectrometer system. These techniques allowed the identification of the reaction mechanisms in each electrode, owing to the analysis of evolved gaseous species, the amount of heat released and mass loss. These results provided insight into the thermal events happening within a broader temperature range than covered in previously published models. This allowed the formulation of an improved thermal model to depict TR. The heat of reaction, activation energy, and frequency factor (thermal triplets) for each major exothermic process at material level were investigated in a Lithium Nickel-Manganese-Cobalt-Oxide (NMC (111))-Graphite battery cell. The results were analyzed, and their kinetics were derived. These data can be used to successfully simulate the experimental heat flow.

This work aims at determining the reaction kinetics of Li-ion battery cathode and anode materials undergoing thermal runaway (TR). Simultaneous Thermal Analysis (STA)/Fourier Transform Infrared (FTIR) spectrometer/Gas Chromatography Mass Spectrometry (GC-MS) were used to reveal thermal events and to detect evolved gases.

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 ...