Top-down ion mobility/mass spectrometry reveals enzyme specificity: Separation and sequencing of isomeric proteoforms.
chromatin
enzyme assay
epigenetics
ion mobility spectrometry
Journal
Proteomics
ISSN: 1615-9861
Titre abrégé: Proteomics
Pays: Germany
ID NLM: 101092707
Informations de publication
Date de publication:
28 Jan 2024
28 Jan 2024
Historique:
revised:
15
11
2023
received:
09
07
2023
accepted:
12
12
2023
medline:
29
1
2024
pubmed:
29
1
2024
entrez:
28
1
2024
Statut:
aheadofprint
Résumé
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-acetylation of residue K16, while also adding 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 separated 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 intact isomeric proteoforms and precise PTM localization.
Identifiants
pubmed: 38282202
doi: 10.1002/pmic.202200471
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2200471Subventions
Organisme : Independent Research Fund Denmark
ID : 0135-00114B
Organisme : Novo Nordisk Foundation
ID : NNF20OC0061575
Organisme : Lundbeck Foundation
ID : R344-2020-1051
Informations de copyright
© 2024 Wiley-VCH GmbH.
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