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&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 (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, ...

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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 ...

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 ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become the most widely used protein purification method in the modern literature. But, the application of affinity chromatography to metal binding proteins, especially those with redox sensitive metals such as iron, is often limited to laboratories with access to a glove box - equipment that is not routinely available in the undergraduate laboratory. In this article, we demonstrate our benchtop methods for isolation, IMAC purification and metal-ion reconstitution of a poly-histidine tagged, redox-active, non-heme iron binding extradiol dioxygenase and the assay of the dioxygenase with varied substrate concentrations and saturating oxygen. These methods are executed by undergraduate students and implemented in the undergraduate teaching and research laboratory with instrumentation that is accessible and affordable at primarily undergraduate institutions.

Here we present a protocol for the benchtop immobilized metal affinity chromatography purification and subsequent reconstitution of a polyhistidine tagged, non-heme iron binding dioxygenase suitable for the undergraduate teaching laboratory.

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

Biochemistry

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 ...

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 ...