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Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry

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    Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry
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    • Bryon S. Drown
      Bryon S. Drown
      Proteomics Center of Excellence, Departments of Molecular Biosciences, Chemistry, and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60202, United States
      More by Bryon S. Drown
      Orcidhttps://orcid.org/0000-0002-5631-512X
    • Raveena Gupta
      Raveena Gupta
      Proteomics Center of Excellence, Departments of Molecular Biosciences, Chemistry, and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60202, United States
      More by Raveena Gupta
    • John P. McGee
      John P. McGee
      Proteomics Center of Excellence, Departments of Molecular Biosciences, Chemistry, and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60202, United States
      More by John P. McGee
    • Michael A. R. Hollas
      Michael A. R. Hollas
      Proteomics Center of Excellence, Departments of Molecular Biosciences, Chemistry, and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60202, United States
      More by Michael A. R. Hollas
      Orcidhttps://orcid.org/0000-0002-0797-3134
    • Paul J. Hergenrother
      Paul J. Hergenrother
      Department of Chemistry, Carl R. Woese Institute for Genomic Biology, Cancer Center at Illinois, University of Illinois at Urbana─Champaign, Urbana, Illinois 61801, United States
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    • Jared O. Kafader
      Jared O. Kafader
      Proteomics Center of Excellence, Departments of Molecular Biosciences, Chemistry, and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60202, United States
      More by Jared O. Kafader
      Orcidhttps://orcid.org/0000-0002-5359-264X
    • Neil L. Kelleher*
      Neil L. Kelleher
      Proteomics Center of Excellence, Departments of Molecular Biosciences, Chemistry, and the Feinberg School of Medicine, Northwestern University, Evanston, Illinois 60202, United States
      *Email: [email protected]
      More by Neil L. Kelleher
      Orcidhttps://orcid.org/0000-0002-8815-3372
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    Analytical Chemistry

    Cite this: Anal. Chem. 2024, 96, 11, 4455–4462
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    https://doi.org/10.1021/acs.analchem.3c04758
    Published March 8, 2024
    Copyright © 2024 American Chemical Society
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    Abstract

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    The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a “bird’s eye” view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0–4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c04758.

    • Validation of MEK1 presence, ion analysis and fragmentation comparisons, and complete fragmentation map of MEK1 coverage (PDF)

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    This article is cited by 2 publications.

    1. You Zou, Che-Fan Huang, Grace R. Sturrock, Neil L. Kelleher, Michael C. Fitzgerald. Top-Down Stability of Proteins from Rates of Oxidation (TD-SPROX) Approach for Measuring Proteoform-Specific Folding Stability. Analytical Chemistry 2024, 96 (49) , 19597-19604. https://doi.org/10.1021/acs.analchem.4c04469
    2. Pei Su, John P. McGee, Michael A. R. Hollas, Ryan T. Fellers, Kenneth R. Durbin, Joseph B. Greer, Bryan P. Early, Ping F. Yip, Vlad Zabrouskov, Kristina Srzentić, Michael W. Senko, Philip D. Compton, Neil L. Kelleher, Jared O. Kafader. Standardized workflow for multiplexed charge detection mass spectrometry on orbitrap analyzers. Nature Protocols 2025, 8 https://doi.org/10.1038/s41596-024-01091-y
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    Analytical Chemistry

    Cite this: Anal. Chem. 2024, 96, 11, 4455–4462
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.3c04758
    Published March 8, 2024
    Copyright © 2024 American Chemical Society
    Request reuse permissions

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