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 voltage into the IM cell) must be checked for their influences on charge-state and ion mobility distributions.
The following are the typical operating conditions that were used in this work. The aqueous peptide samples were injected using a blunt nose 1.0 mL syringe using a 10 &#956;L min&#8722;1 flow rate, 2.0 kV capillary voltage for positive ions (+) or &#8722;1.8 kV for negative ions (-), 130 &#176;C source temperature, 250 &#176;C desolvation, 20 V sampling cone, and 4.0 V extraction cone. The IM section was operated with 6.0 V entrance voltage to the trap cell with an argon pressure of 2.25 x 10&#8722;2 mbar using a 1.5 mL/min flow rate. The voltage for injecting ions (trap DC bias) into the IM cell was set at 12 V to avoid dissociation of ions as they initially collided with the nitrogen buffer gas. The IM cell separated ions based on their charge and collision cross-section and utilized a 0.52 mbar nitrogen pressure and 20.0 mL min&#8722;1 flow rate. The IM was operated with ramped 12.0&#8211;20.0 V (+) or 8.0&#8211;30.0 V (&#8722;) travelling wave heights and ramped 800&#8211;1,500 m s&#8722;1 (+) or 250&#8211;1,000 m s&#8722;1 (&#8722;) velocities for every sweep through the cell of the IM travelling wave. The transfer cell was operated with the same argon pressure as the trap cell and guided the IM resolved ions to the orthogonal time-of-flight mass-to-charge analyzer. The ion mobility-mass spectra were acquired by synchronizing the gated release of ions into the IM cell with the time-of-flight mass-to-charge analyzer.
Using native ESI conditions, solution-phase properties such as the charge state and conformational state are conserved during the IM-MS analyses. For example, the charge states of mb-OB3b and ambs observed during IM-MS analyses20,37 were closely related to the charge states expected in the solution phase40. The mb-OB3b peptide is tetraprotic and forms only negatively charged ions during IM-MS analysis40, whether Cu(I)-bound or Cu(I)-free, because it contains the C-terminus (pKa &#60; 1.7), two enethiol oxazolone groups (pKa = 5.0 and 9.7), and Tyr group (pKa = 11.0)42. The ambs in their fully protonated form will have an overall charge of +2 because of the C-terminus (pKa &#8776; 2), two His (pKa = 6.0), two Cys (pKa = 8.3), and Tyr (pKa = 11.0) sites19,41. Thus, they generally form positively charged ions at a pH of &#60;6 and negatively charged ions at a pH of &#62;6.
The ambs also showed clear pH-dependent Cu(I/II) binding behavior and redox activity in which Cu(I)-binding at a low pH transitioned to Cu(II) binding at a higher pH. The Cu(I/II) reactions included forming the oxidized amb species (ambox) that contained disulfide bonds and various multimers and multiple Cu(I/II) binding (Figure 5 and Figure 6). These redox reactions are time-dependent and it was shown that the longer the time interval (up to 210 min) between sample preparation and IM-MS analyses the more oxidized products were observed37. Therefore, careful consideration of reaction time dependence on the observation of products is also required.
Limitations: IM-MS and theoretical collision cross-sections identify which type of coordination each metal ion prefers 
To help interpret the IM-MS m/z and CCS data, an extensive search was conducted using the B3LYP/LanL2DZ level of theory. Geometry-optimized conformers with different coordination sites were compared between their predicted free energy and agreement with the CCS measured by IM-MS. Molecular modeling of these peptides and their complexes is limited by the type of electronic structure calculations that can be applied to these relatively large systems. Other methods that have been studied or recommended include work by Truhlar et al.43, who found that M05-2X was the best DFT functional and PM7 and MNDO/d were good NDDO semi-empirical methods for Zn(II)-containing compounds44. These peptides have a large conformational space and thorough investigation to locate the lowest energy conformers must include comparing the various metal chelating sites, various cis- and trans-peptide bonds, salt-bridges, hydrogen bonding, and &#960;-cation interaction between the aromatic Tyr side group and metal cation.
Significance with respect to existing methods: Cu(I/II) and other selected metal ion binding compared between mb-OB3b and ambs 
X-ray crystallography and NMR spectroscopy are the most common techniques used for determining the atomic resolution of peptides tertiary structure. However, X-ray crystallography studies of metallopeptides are scarce due to problems with the crystallization of these complexes45. NMR is also not suitable for the interpretation of a sample where closely related individual oligopeptide species are present46. Therefore, IM-MS and DFT molecular modelling are alternative techniques for studying peptide reactions especially those that result from complex redox and Cu(I/II)-binding reactions20,37,40,47. The strength of IM-MS is that it can resolve each of the products and identify their molecular composition by simultaneously measuring their m/z and arrival times that relate to the stoichiometry, protonation state, and conformational structure.
For example, the mb-OB3b will chelate a variety of metal ions, and its selectivity towards each ion was displayed by the IM-MS metal ion titrations (Figure 7). The results showed the mb-OB3b preference for binding Cu(I) and Ag(I), while comparing the results at a pH of 7 with amb7. Figure 8 shows amb7 preferentially chelates Zn(II) and Ni(II). In general, the amb studies showed that replacing the two enethiol-oxazolones with 2His-2Cys did not exclude Cu(I/II)-binding, but it resulted in multiple Cu(I)-binding via linear-bridging coordination (Figure 3) as opposed to the mononuclear Cu(I) binding of mb-OB3b&#8217;s tetrahedral coordination48. Cu(II) reduction was also mediated by thiol oxidation and disulfide bridge formation in contrast to the existing disulfide bridge in apo-mb-OB3b and the high reduction potential for copper-loaded mb-OB3b, which supports the strong preference for Cu(I)49.
Future applications 
Further IM-MS studies of amb peptides are underway, in which their primary sequence is modified by replacing the His or Cys with Gly or Asp, while the Tyr residue is replaced with either Gly or Phe. These studies are also being conducted in 10.0 mM ammonium acetate, with the pH modified with ammonium hydroxide (for pH = 7, 8, and 9) to keep the total ionic strength constant for each sample. These results will be published shortly.

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&amp;amp;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 (amb1ox) by disulfide bridge formation, and at a pH of &#62;6, the [amb1ox&#8722;3H+Cu(II)]&#8722; ion (Figure 2A) was formed. This indicated the deprotonation of two imidazoliums, carboxyl group, and two additional sites that were coordinating Cu(II).
Molecular modeling of the [amb1ox&#8722;3H+Cu(II)]&#8722; ion using B3LYP/LanL2DZ determined the lowest energy complex was Cu(II) coordinated via the imidazole &#948;N of His1 and the deprotonated nitrogens of the backbone amide groups of Cys2 and Gly3. However, below a pH of 6, adding Cu(II) to amb1 formed a m/z isotope pattern that could only be accounted for by Cu(I) binding, forming the [amb1ox+Cu(I)]+ ion (Figure 2B). In contrast, a pH higher than 6 caused the m/z isotope pattern to decrease 1 m/z, accounted for by the positive charged [amb1ox&#8722;H+Cu(II)]+ ion. Adding Zn(II) did not oxidize amb1, and Zn(II) binding was observed at a pH of &#62;6, primarily forming the [amb1&#8722;3H+Zn(II)]&#8722; ion (Figure 2C). This indicated the deprotonation of the imidazoliums, thiol, and carboxyl groups. Molecular modeling of the [amb1&#8722;3H+Zn(II)]&#8722; ion determined the lowest energy conformers to be either tetrahedral Zn(II) coordination via 2His-2Cys or His-2Cys and the carboxylate of the C-terminus.
Multiple Cu(I) binding of amb2 
The redox reactions between Cu(II) and amb2 (Figure 1B) resulted in Cu(I) binding. This was studied in more detail using IM-MS, UV-Vis spectrophotometry, and B3LYP molecular modeling37. The main products of the Cu(II) titration of amb2 at a pH of 5 were amb2 oxidation (through disulfide bridge formation) and the unoxidized amb2 species coordinating three Cu(I) ions.
A search using the B3LYP/LanL2DZ method located two low-energy complexes contending for the 3Cu(I) coordinated species. The first was the complex shown in Figure 3A, where the 3Cu(I) ions were coordinated via the bridging thiolate groups38 of Cys2 and Cys6 (of His1) as well as &#948;N1 and &#948;N5 (of His5). The second complex (3c) has a salt bridge between the protonated His1 side group and C-terminal carboxylate group. These results suggest that at a pH of 3.0&#8211;6.0, the principal amb2+3Cu(I) complex is the salt-bridged structure, which can be successfully transferred from solution to gas-phase with only minimal structural rearrangement.
The theoretical LJ CCS of 209 &#177; 6 &#197;2, calculated using the Sigma program36 for complex 3c, agreed with the IM-MS measured CCS, indicating that 3c represents [amb2&#8722;2H+3Cu(I)]+ conformation at pH 3.0-6.0. However, at a pH of &#62;6, this complex was not observed by IM-MS, probably because further deprotonation of His1 (pKa= 6.0) results in an overall neutral complex. Once the imidazoleum group of His1 is deprotonated, 3Cu(I) coordination may convert to the bridging thiolate groups of Cys2 and Cys6 as well as &#948;N1 and &#948;N5 of His1 and His5, respectively (3a).
The pH dependence of amb4 Cu(I/II)-binding and redox activity 
The IM-MS and B3LYP techniques have been used to investigate the Cu(II) and pH titrations of amb4 (Figure 1C) and identified monomer, dimer, trimer, and tetramer complexes of amb4 containing up to three Cu(I) ions or two Cu(II) ions for each monomer subunit39. The complexes also contained various numbers of disulfide bridges, and these products were produced whether or not the Cu(II) reactions with amb4 were conducted in anaerobic or aerobic aqueous solutions.
Using the IM-MS technique, it was shown that these individual species could be separated and quantified even if they had overlapping isotope patterns because of differences in their arrival times (Figure 4). The identification and quantification of these closely related species is a task that no other instrumental or analytical technique can achieve. These IM-MS studies provide considerable insight into the pH-dependent redox reactions and exactly identified the numbers of inter- or intra-molecular disulfide bridges, number of Cu(I) or Cu(II) ions, and number of deprotonation sites in each of the complexes (Figure 5).
Moreover, measuring the complexes CCS also allowed the determination of each of the individual species conformational size, which was used with an extensive B3LYP/LanL2DZ search to locate conformers with structures that agreed with both the correct molecular stoichiometry and CCS measured by IM-MS. Through this method, the Cu(I/II) coordination of the various complexes were identified. The reactions between Cu(II) and amb4 included the formation of dimers, trimers, and tetramers coordinating either Cu(I) or Cu(II), depending on the pH of the solution.
For example, in solutions that were mildly acidic (pH = 3.0&#8211;6.0), they primarily bound Cu(I) ions and were unoxidized, while in solutions that were slightly basic (pH = 8.0&#8211;11.0), they primarily bound Cu(II) ions and were oxidized by all the Cys forming disulfide bonds (Figure 6). The B3LYP/LanL2DZ determined that the Cu(I) ions were linear and bridged by the thiolate and imidazole groups, while the Cu(II) ions were chelated via distorted T-shaped or square planar geometries by an imidazole as well as the deprotonated backbone nitrogens of amide groups.
IM-MS analysis of mb-OB3b 
The IM-MS studies19,40 of mb-OB3b (Figure 1D) showed that in the gas-phase, Cu(I)-free mb-OB3b exists as three negatively charged species: [mb-OB3b&#8211;H]&#8211;, [mb-OB3b&#8211;2H]2&#8211;, and [mb-OB3b&#8211;3H]3&#8211;, consistent with expected solution-phase behavior. Individual metal ion titrations were performed19 to determine the metal ion selectivity of mb-OB3b. Figure 7 shows the results of the selected metal ion titrations and shows that the apparent binding selectivity of mb-OB3b can be categorized as three major groups: 1) Cu(I) and Ag(I); 2) Ni(II), Zn(II) and Co(II); and 3) Pb(II), Fe(II), and Mn(II). This order of binding selectivity was shown to be in general agreement with that found by fluorescence quenching experiments19 and isothermal titration calorimetry18.
Comparison of mb-OB3b and amb7 metal binding selectivity 
The apparent binding selectivity of mb-OB3b was compared to the binding selectivity of amb7 at a pH of 7. The amb7 was designed with the same amino acid sequence as mb-OB3b, but with the two enethiol oxazolone groups replaced with two His-Cys groups. The amb7 (Figure 1E) has a single disulfide bond between Cys6 and Cys12. The results of the formation of negative charged complexes (Figure 8) showed that amb7 preferred binding selectivity for Ni(II) and Zn(II) (60%), followed by Co(II) and Pb(II) (40%). Furthermore, there was about 20% Cu(II) binding. There was either trace or no amb7 binding of Ag(I), Mn(II), or Fe(II). This compared to mb-OB3b&#8217;s preferred binding selectivity of over 90% for Cu(I) and Ag(I) binding.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60102/60102fig01.jpg" /> 
Figure 1: Primary structures of the alternative methanobactin (amb) and methanobactin (mb-OB3b) peptides.&#160;(A) Acetyl-His1-Cys2-Gly3-Pro4-His5-Cys6 (amb1); (B) acetyl-His1-Cys2-Tyr3-Pro4-His5-Cys6 (amb2); (C) acetyl-His1-Cys2-Gly3-Ser4-Tyr5-Pro6-His7-Cys8-Ser9 (amb4); (D) 1-(N-[mercapto-(5-oxo-2-(3-methylbutanoyl)oxazol-(Z)-4-ylidene)methyl]-Gly1&#8211;Ser2&#8211;Cys3&#8211;Tyr4)-pyrrolidin-2-yl-(mercapto-[5-oxo-oxazol-(Z)-4-ylidene]methyl)&#8211;Ser5&#8211;Cys6&#8211;Met7 (mb-OB3b); and (E) acetyl-Leu1-His2-Cys3-Gly4-Ser5-Cys6-Tyr7-Pro8-His9-Cys10-Ser11-Cys12-Met13 (amb7). Shading shows the: 2His-2Cys or enethiol-oxazolone binding sites (<img alt="Icon" src="/files/ftp_upload/60102/pink.jpg" />); proline or pyrrolidine hinges (<img alt="Icon" src="/files/ftp_upload/60102/blue.jpg" />); acetyl or methylbutanol group N-terminus (<img alt="Icon" src="/files/ftp_upload/60102/green.jpg" />); and tyrosine, which can stabilize metal ion coordination via a second solvation shell &#960;&#8211;cation interaction (<img alt="Icon" src="/files/ftp_upload/60102/yellow.jpg" />). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60102/60102fig02.jpg" /> 
Figure 2: Mean relative intensities of the alternative methanobactin (amb1) acetyl-His1-Cys2-Gly3-Pro4-His5-Cys6 and metal-bound complex (amb1+X) (where X = Cu or Zn).&#160;Observations were made during negative and positive ion mass spectrometry analyses of 1:1 molar ratio solution of amb:XCl2 over the pH range of 3.0&#8211;11.0. Error bars show standard deviations of the means of both the relative intensity and pH from three replicate pH titration experiments. The 1:1 molar solution of amb:CuCl2 resulted in the oxidation of amb (ambox) with Cys2 and Cys6, forming a disulfide bridge. (A) Negative ion analysis of amb:CuCl2 showing [ambox&#8722;H]&#8722; and [ambox&#8722;3H+Cu(II)]&#8722;. (B) Positive ion analysis of amb:CuCl2 showing [ambox]+ and [ambox+Cu(I/II)]+; the oxidation state of Cu in the complex was pH-dependent, being [ambox+Cu(I)]+;&#160; below a pH of 8 and [ambox&#8722;H+Cu(II)]+;&#160;above a pH of 8. (C) Negative ion analysis of amb:ZnCl2 showing [amb]n&#8722; and [amb+Zn(II)]n&#8722;. (D) Positive ion analysis of amb:ZnCl2 showing [amb]n+ and [amb+Zn(II)]n+. This figure has been adapted from a previous publication20. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60102/60102fig03.jpg" /> 
Figure 3: Proposed structures of [amb2+3Cu(I)]+ using lowest energy and geometry-optimized structures located from the B3LYP/LanL2DZ level of theory.&#160;(A) 3 Cu(I) coordination via &#948;N1&#948;N5 of His1 and His5 and thiolate bridging thiolate groups of Cys2 and Cys6 with a theoretical cross-section of 217 &#177; 6 &#197;2. (B) Illustration of the &#948;N1&#948;N5 and thiolate bridging coordination. (C) Salt bridged structure showing the 3 Cu(I) coordination via carboxylate terminal (Cys6), &#948;N5, and thiolate bridging with a theoretical cross section of 209 &#177; 6 &#197;2. (D) Illustration of the carboxylate terminal, &#948;N5, and thiolate bridging coordination. Bonding distances A, B, C, D, E, and F are shown in the unit of &#197;. This figure has been adapted from a previous publication37. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60102/60102fig04.jpg" /> 
Figure 4: IM-MS analysis of products of the 1:1 mixture of amb4:Cu(II) at pH = 4.4.&#160;(A) Extracted isotope patterns for the [amb4&#8722;2H+3Cu(I)]+, [diamb4&#8722;4H+6Cu(I)]2+, [triamb4&#8722;6H+9Cu(I)]3+ and [tetraamb4&#8722;8H+12Cu(I)]4+ species. (B) Integration of the extracted arrival times of [amb4&#8722;2H+3Cu(I)]+, [diamb4&#8722;4H+6Cu(I)]2+, [triamb4&#8722;6H+9Cu(I)]3+ and [tetraamb4&#8722;8H+12Cu(I)]4+ were used to calculate their relative intensities. To calculate the percent relative intensities, the summation of the integrated area for all extracted species for each titration point was used to normalize to the percent scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60102/60102fig05.jpg" /> 
Figure 5: Changing isotope pattern for singly Cu(I/II) bound amb4 observed during the pH titration of molar equivalents of Cu(II):amb4 at pH = 4.04, 6.02, and 9.98.&#160;At pH = 4.04, the experimental result primarily matches the isotope model for [amb4+Cu(I)]+. At pH = 6.02, there is a shift of -2 m/z, signifying the formation of the disulfide bridge (shown as oxidation of amb4ox) and agreement with the isotope pattern for [amb4ox+Cu(I)]+. At pH = 9.98, there is a further shift of -1 m/z, signifying Cu(II) binding and the removal of a proton to maintain the +1 charge state, which then matches the isotope pattern for [amb4ox&#8722;H+Cu(II)]+. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60102/60102fig06.jpg" /> 
Figure 6: Changing relative intensities of identities of the Cu(I/II) complexes of the monomer, dimer, and trimer of amb4 over pH range of 3.0&#8211;11.0.&#160;(A) Monomer with one Cu(I/II) ion, (B) dimer with 2 Cu(I/II) ions, and (C) trimer with 3 Cu(I/II) ions. The captions note how many disulfide bonds were present in the complex. This figure has been adapted from a previous publication39. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60102/60102fig07.jpg" /> 
Figure 7: Percentage of formation of the Cu(I), Ag(I), Zn(II), Ni(II), Co(II), Mn(II), Pb(II), or Fe(II) complexes of methanobactin.&#160;Observed during the individual metal ion titrations of methanobactin. It should be noted that Cu(I) binding resulted from the addition of Cu(II) and Fe(II) binding from the addition of Fe(III). This figure has been adapted from a previous publication19. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60102/60102fig08.jpg" /> 
Figure 8: Comparison of the percentage of Cu(I/II), Ag(I), Zn(II), Ni(II), Co(II), Mn(II), Pb(II), or Fe(II) chelation by mb-OB3b and amb7 at pH = 7.&#160;The comparison is for the formation of negatively charged ions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/60102fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary File" src="/files/ftp_upload/60102/gauss.jpg" /> 
Supplementary File. GaussView usage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60102/GaussView-SI.docx" target="_blank">Please click here to download this file.</a>

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, ...

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9.1K Views.  Texas A&M University-Commerce. Ion mobiliteitsmassaspectrometrie, of IM-MS, identificeert de verschillende producten ionen van de pH-afhankelijke redox en methylbindende reactie van peptiden. Met de moleculaire modellering van hun tertiaire structuur kan de metaalcorrelatie worden bepaald. IM-MS kan elk van de productionen oplossen en hun moleculaire samenstelling identificeren door tegelijkertijd hun massa-to-charge en aankomsttijden te meten en met betrekking tot hun stoichiometrie, protonatietoestand en conformatiestruc...

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 ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

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 ...

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

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)]-&#160;+ 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&#39; 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 (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

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.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Biology

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been reported to directly bind to a variety of peptides and play both necessary and pathological roles ranging from mediating structure to electron transfer properties to imparting catalytic function. Quantifying the binding affinity and thermodynamics of these Cu(II)-peptide complexes in vitro provides insight into the thermodynamic driving force of binding, potential competitions between different metal ions for the peptide or between different peptides for Cu(II), and the prevalence of the Cu(II)-peptide complex in vivo. However, quantifying the binding thermodynamics can be challenging due to a myriad of factors, including accounting for all competing equilibria within a titration experiment, especially in cases where there are a lack of discrete spectroscopic handles representing the peptide, the d-block metal ion, and their interactions.
Here, a robust set of experiments is provided for the accurate quantification of Cu(II)-peptide thermodynamics. This article focuses on the use of electronic absorption spectroscopy in the presence and absence of chromophoric ligands to provide the needed spectroscopic handle on Cu(II) and the use of label-free isothermal titration calorimetry. In both experimental techniques, a process is described to account for all competing equilibria. While the focus of this article is on Cu(II), the described set of experiments can apply beyond Cu(II)-peptide interactions, and provide a framework for accurate quantification of other metal-peptide systems under physiologically relevant conditions.

Quantifying the Binding Interactions Between CuII and Peptide Residues in the Presence and Absence of Chromophores

This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

Copper(II) is an essential metal in biological systems, conferring unique chemical properties to the biomolecules with which it interacts. It has been ...

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

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