Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies

Simone Sidoli, Benjamin A. Garcia

Research output: Chapter in Book/Report/Conference proceedingChapter

18 Scopus citations

Abstract

Histone posttranslational modifications (PTMs) play an essential role in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. Such PTMs are mostly localized on histone N-terminal tails where, as single units or in a combinatorial manner, they infl uence chromatin reader protein binding and fine-tune the abovementioned activities. Mass spectrometry (MS) is currently the most adopted strategy to characterize proteins and protein PTMs. We hereby describe the protocols to identify and quantify histone PTMs and their patterns using either bottom-up or middle-down proteomics. In the bottom-up strategy we obtain 5–20 aa peptides by derivatization with propionylation followed by trypsin digestion. The newly generated N-termini of histone peptides can be further derivatized with light or isotopically heavy propionyl groups to increase chromatographic retention and allow multiplexed analyses. Moreover, we describe how to perform derivatization and trypsin digestion of histones loaded into a gel, which is usually the final step of immunoprecipitation experiments. In the middle-down strategy we obtain intact histone tails of 50–60 aa by digestion with the enzyme GluC. This allows characterization of combinatorial histone PTMs on N-terminal tails.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages121-148
Number of pages28
DOIs
StatePublished - Jan 1 2017
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1528
ISSN (Print)1064-3745

Keywords

  • Bottom-up
  • Histones
  • Mass spectrometry
  • Middle-down
  • Proteomics

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Sidoli, S., & Garcia, B. A. (2017). Characterization of individual histone posttranslational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies. In Methods in Molecular Biology (pp. 121-148). (Methods in Molecular Biology; Vol. 1528). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6630-1_8