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Related Concept Videos

Electrospray Ionization (ESI) Mass Spectrometry01:12

Electrospray Ionization (ESI) Mass Spectrometry

Higher molecular weight biomolecules are nonvolatile compounds that may decompose before ionizing or vaporizing during mass analysis with conventional electron impact ionization methods. Accordingly, electrospray ionization (ESI) is the favored method for vaporizing and ionizing biomolecules as it circumvents rapid fragmentation and enables the recording of mass signals for the entire biomolecule.
ESI utilizes electrical energy to transfer ions from the liquid phase of the sample into the...

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Related Experiment Video

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Analyzing Large Protein Complexes by Structural Mass Spectrometry
15:35

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Published on: June 19, 2010

Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry.

Amr El-Hawiet, Elena N Kitova, Denis Arutyunov

    Analytical Chemistry
    |April 18, 2012
    PubMed
    Summary

    A new proxy protein electrospray ionization mass spectrometry (ESI-MS) method quantifies protein-ligand interactions. This technique uses a detectable proxy protein to measure binding affinities for complexes not directly observable by ESI-MS.

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    Last Updated: May 23, 2026

    Analyzing Large Protein Complexes by Structural Mass Spectrometry
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    Analyzing Large Protein Complexes by Structural Mass Spectrometry

    Published on: June 19, 2010

    Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry
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    Published on: October 15, 2018

    Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies
    10:01

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

    Published on: November 28, 2017

    Area of Science:

    • Biochemistry
    • Analytical Chemistry
    • Mass Spectrometry

    Background:

    • Direct quantification of protein-ligand complexes using electrospray ionization mass spectrometry (ESI-MS) is challenging for complexes not directly detectable.
    • A novel proxy protein ESI-MS method is introduced, combining direct ESI-MS measurements with competitive binding assays.

    Discussion:

    • The method utilizes a proxy protein (P(proxy)) with known affinity for the ligand, which is directly detectable by ESI-MS, to indirectly quantify binding to the target protein.
    • A mathematical framework is provided for determining the association constant (K(a)) using this method, including a modified approach for real-time ligand concentration changes.

    Key Insights:

    • The proxy protein ESI-MS method was validated for interactions between bacteriophage P22 tailspike proteins and Salmonella O-antigen fragments (octasaccharide and dodecasaccharide).
    • A 27 kDa single-chain antibody served as an effective proxy protein, demonstrating the method's reliability.
    • Binding affinity measurements at 10 and 25 °C showed excellent agreement with results from a fluorescence quenching assay.

    Outlook:

    • This method offers a powerful tool for quantifying challenging protein-ligand interactions in various biological systems.
    • Further applications could involve studying transient complexes or those with low affinities not amenable to traditional methods.
    • The development expands the utility of mass spectrometry in quantitative proteomics and drug discovery.