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Top-down ion mobility/mass spectrometry reveals enzyme specificity: Separation and sequencing of isomeric proteoforms
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  • Francis Berthias,
  • Nurgül Bilgin,
  • Jasmin Mecinovic,
  • Ole Jensen
Francis Berthias
University of Southern Denmark
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Nurgül Bilgin
University of Southern Denmark
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Jasmin Mecinovic
University of Southern Denmark
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Ole Jensen
University of Southern Denmark

Corresponding Author:[email protected]

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Enzymatic catalysis is one of the fundamental processes that drives the dynamic landscape of post-translational modifications (PTMs), expanding the structural and functional diversity of proteins. Here, we assessed enzyme specificity using a top-down ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS) workflow. We successfully applied trapped IMS (TIMS) to investigate site-specific N-ε-acetylation of lysine residues of full-length histone H4 catalyzed by histone lysine acetyltransferase KAT8. We demonstrate that KAT8 exhibits a preference for N-ε-actylation of residue K16, while also installing N-ε-acetyl groups on residues K5 and K8 as the first degree of acetylation. Achieving TIMS resolving power values of up to 300, we fully separated mono-acetylated regioisomers (H4K5ac, H4K8ac, and H4K16ac). Each of these regioisomers produce unique MS/MS fragment ions, enabling estimation of their individual mobility distributions and the exact localization of the N-ε-acetylation sites. This study highlights the potential of top-down TIMS-MS/MS for conducting enzymatic assays at the intact protein level and, more generally, for separation and identification of isomeric proteoforms and precise PTM localization.
09 Jul 2023Submitted to PROTEOMICS
11 Jul 2023Assigned to Editor
11 Jul 2023Review(s) Completed, Editorial Evaluation Pending
11 Jul 2023Submission Checks Completed
11 Jul 2023Reviewer(s) Assigned
11 Sep 2023Editorial Decision: Revise Minor
13 Nov 2023Review(s) Completed, Editorial Evaluation Pending
13 Nov 20231st Revision Received
16 Nov 2023Reviewer(s) Assigned