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Related Concept Videos

Histone Modification02:32

Histone Modification

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The histone proteins in the nucleosomes are post-translationally modified (PTM) to increase or decrease access to DNA. The commonly observed PTMs are methylation, acetylation, phosphorylation, and ubiquitination of lysine amino acids in the histone H3 tail region. These histone modifications have specific meaning for the cell. Hence, they are called "histone code". The protein complex involved in histone modification is termed as "reader-writer" complex.
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Related Experiment Video

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The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
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A Super-SILAC Approach for Profiling Histone Posttranslational Modifications.

Roberta Noberini1, Elisa Longhi2, Tiziana Bonaldi3

  • 1Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy. Roberta.noberini@ieo.it.

Methods in Molecular Biology (Clifton, N.J.)
|November 12, 2022
PubMed
Summary

Super-SILAC, an advanced method, accurately quantifies histone posttranslational modifications (PTMs) using mass spectrometry. This technique overcomes limitations of traditional methods, enabling broader applications in biological research.

Keywords:
EpigeneticsHistone posttranslational modificationsMass spectrometrySuper-SILAC

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Histone posttranslational modifications (PTMs) are crucial regulators of gene expression with roles in health and disease.
  • Mass spectrometry (MS) is the leading technology for quantifying histone PTMs.
  • Stable-isotope labeling by amino acids in cell culture (SILAC) offers accurate quantitation but has limitations.

Purpose of the Study:

  • To present an optimized protocol for generating a histone-focused super-SILAC mix.
  • To utilize super-SILAC as an internal standard for precise histone PTM quantitation.

Main Methods:

  • Development of a super-SILAC spike-in standard using a mix of heavy-labeled cell lines.
  • Application of the super-SILAC standard for quantitative analysis of histone PTMs via mass spectrometry.

Main Results:

  • Super-SILAC overcomes the limited multiplexing and cell division requirements of classical SILAC.
  • This method enhances quantitation accuracy and reduces experimental variability in histone PTM analysis.

Conclusions:

  • Super-SILAC provides a robust and versatile approach for accurate histone PTM quantitation.
  • The described protocol facilitates the application of super-SILAC in diverse research settings, including clinical samples.