Identification and Quantification of Histone PTMs Using High-Resolution Mass Spectrometry

K. R. Karch, S. Sidoli, B. A. Garcia

Research output: Chapter in Book/Report/Conference proceedingChapter

10 Scopus citations

Abstract

DNA is organized into nucleosomes, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins including H2A, H2B, H3, and H4. Histones are critical regulators of many nuclear processes, including transcription, DNA damage repair, and higher order chromatin structure. Much of their function is mediated through extensive and dynamic posttranslational modification (PTM) by nuclear enzymes. Histone PTMs are thought to form a code, where combinations of PTMs are responsible for specific biological functions. Here, we present protocols to identify and quantify histone PTMs using nanoflow liquid chromatography coupled to mass spectrometry (MS). We first describe how to purify histones and prepare them for MS. We then describe three MS platforms for histone PTM analysis, including bottom-up, middle-down, and top-down approaches, and explain the relative benefits and pitfalls of each approach. We also include tips to increase the throughput of large experiments.

Original languageEnglish (US)
Title of host publicationEnzymes of Epigenetics, Part B
EditorsRonen Marmorstein
PublisherAcademic Press Inc.
Pages3-29
Number of pages27
DOIs
StatePublished - 2016
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume574
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Bottom-up
  • Epigenetics
  • Histone
  • Mass spectrometry
  • Middle-down
  • Posttranslational modifications
  • Top-down

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Karch, K. R., Sidoli, S., & Garcia, B. A. (2016). Identification and Quantification of Histone PTMs Using High-Resolution Mass Spectrometry. In R. Marmorstein (Ed.), Enzymes of Epigenetics, Part B (pp. 3-29). (Methods in Enzymology; Vol. 574). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2015.12.007