Name: Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides (Video) | JoVE Journal
Uploaded: 2019-09-07T15:00:00.000+00:00
Duration: 11 min 4 s
Description: Discover ion mobility-mass spectrometry techniques for analyzing metal-binding peptides and their therapeutic potential. This method combines IM-MS with molecular modeling to characterize peptide structures.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 °C until use.</li>
	<li>Synthesize the amb peptides (&gt;98% purity for amb1, amb2, amb4; &gt;70% purity for amb7), freeze-dry the samples, and store them at -80 °C until use.</li>
	<li>Purchase &gt;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 10.0–20.0 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 (&gt;17.8 MΩ 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 μL aliquots from the peptide sample into individually labelled 1.5 mL vials and store them at -80 °C until use.</li>
	</ol>
	</li>
	<li>Metal ion stock solutions
	<ol>
		<li>Weigh accurately, using at least three significant figures, the mass of 10.0–30.0 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 μL 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 μL 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 μL 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 μL aliquots, and place each into a 1.7 mL vial and store at -80 °C.</li>
</ol>
3. Electrospray-ion mobility-mass spectrometry analysis
<ol>
	<li>Clean the ESI entrance tubing and needle capillary thoroughly with about 500 μL of 0.1 M glacial acetic acid, 0.1 M ammonium hydroxide, and finally DI water.</li>
	<li>Thaw a 50.0 μL aliquot of the 1,000 ppm PA stock solution and dilute it with 450 μL of DI water to give a 100 ppm PA. Pipet 100.0 μL of this solution and dilute it to 1.00 mL with 500 μL of DI water and 500 μL 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 μL 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 μL 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 μL of the 0.125 mM amb into a 1.7 mL vial, dilute with 500 μL of DI water, and mix the solution thoroughly.</li>
	<li>Adjust the pH of the sample to 3.0 by adding 50 μL of 1.0 M acetic acid solution.</li>
	<li>Add 200.0 μL 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 μL of the sample and collect the negative and positive ion ES-IM-MS spectra for 5 min each. Use the remaining 500 μL of the sample to record its final pH using a calibrated micro pH electrode.</li>
	<li>Repeat steps 3.6–3.9, 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 3.1–3.5.</li>
	<li>Pipet 200.0 μL of the 0.125 mM amb into a 1.7 mL vial, dilute with 500.0 μL of DI water and mix the solution thoroughly.</li>
	<li>Adjust the pH of the sample to pH = 9.0 by adding 80 μL of the 0.010 M ammonium hydroxide solution.</li>
	<li>Add 28 μL 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 μL of the sample and collect the negative and positive ion ESI-IM-MS spectra for 5 min each. Use the remaining 500 μL of the sample to record its final pH using a calibrated micro pH electrode.</li>
	<li>Repeat steps 4.2–4.5, 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’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 (Ω) of PA negative25,26 and positive27 ions measured in He buffer gas28 to corrected CCS (Ωc) using Equation 1 below, where: z = ion charge; ec = electron charge (1.602×10-19 C); mN2 = mass of N2 gas (Da); and mion = ion mass.29</li>
</ol>
<img alt="Cyclotron frequency equation, formula for particle motion in electromagnetic fields, equation." src="/files/ftp_upload/60102/60102equ01.jpg" data-altupdatedat="1788282455000" data-alt="Equation 1">
<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="Time-of-flight mass spectrometry formula for ion drift time calculation." src="/files/ftp_upload/60102/60102equ02.jpg" data-altupdatedat="1788282455000" data-alt="Equation 2">
<ol start="3">
	<li>Plot the PA calibrants’ tD vs. their Ωc. Then, using a least-squares regression fit of Equation 3 shown below, determine the A' and B values, where: A' 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 for decay rate constant, Omega = A' * t^B, in decay rate analysis research." src="/files/ftp_upload/60102/60102equ03.jpg" data-altupdatedat="1788282455000" data-alt="Equation 3">
<ol start="4">
	<li>Using these A' and B values and the centroid tD value from the ATD of the ambs determine their Ωc using Equation 3 and their Ω 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

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 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</...

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

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

Research

JoVE Journal

Chemistry

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.

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

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.

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

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.

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

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

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

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.

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

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, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

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.

High Resolution Physical Characterization of Single Metallic Nanoparticles

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.

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

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.

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

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.

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

Discover ion mobility-mass spectrometry techniques for analyzing metal-binding peptides and their therapeutic potential. This method combines IM-MS with molecular modeling to characterize peptide structures.

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides (Video) | JoVE Journal

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

Explore More Videos