Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry

Muhammad Mukram Mohamed Mackeen, Holger B. Kramer, Kai Hsuan Chang, Matthew L. Coleman, Richard J. Hopkinson, Christopher J. Schofield, Benedikt M. Kessler

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation, as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultraperformance liquid chromatography high/low collision switching (UPLC-MSE) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3, following a simple chemical derivatization procedure with d6-acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.

Original languageEnglish
Pages (from-to)4082-4092
Number of pages11
JournalJournal of Proteome Research
Volume9
Issue number8
DOIs
Publication statusPublished - 6 Aug 2010
Externally publishedYes

Fingerprint

Histone Code
Histones
Mass spectrometry
Mass Spectrometry
Molecules
Post Translational Protein Processing
Lysine
Cell Growth Processes
Histone Demethylases
HEK293 Cells
Gene Expression Regulation
Pathologic Processes
Autoimmunity
Immunoblotting
Liquid Chromatography
Chromatin
Cell Differentiation
Esters
Liquid chromatography
Cell growth

Keywords

  • 2-oxoglutarate oxygenases
  • Demethylases
  • epigenetics
  • histone demethylation
  • JMJD2A
  • mass spectrometry
  • post-translational modifications
  • proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Mohamed Mackeen, M. M., Kramer, H. B., Chang, K. H., Coleman, M. L., Hopkinson, R. J., Schofield, C. J., & Kessler, B. M. (2010). Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry. Journal of Proteome Research, 9(8), 4082-4092. https://doi.org/10.1021/pr100269b

Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry. / Mohamed Mackeen, Muhammad Mukram; Kramer, Holger B.; Chang, Kai Hsuan; Coleman, Matthew L.; Hopkinson, Richard J.; Schofield, Christopher J.; Kessler, Benedikt M.

In: Journal of Proteome Research, Vol. 9, No. 8, 06.08.2010, p. 4082-4092.

Research output: Contribution to journalArticle

Mohamed Mackeen, Muhammad Mukram ; Kramer, Holger B. ; Chang, Kai Hsuan ; Coleman, Matthew L. ; Hopkinson, Richard J. ; Schofield, Christopher J. ; Kessler, Benedikt M. / Small-molecule-based inhibition of histone demethylation in cells assessed by quantitative mass spectrometry. In: Journal of Proteome Research. 2010 ; Vol. 9, No. 8. pp. 4082-4092.
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