SILAC-based quantitative strategies for accurate histone posttranslational modification profiling across multiple biological samples

Alessandro Cuomo, Monica Soldi, Tiziana Bonaldi

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented “shotgun” workfl ow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages97-119
Number of pages23
Volume1528
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1528
ISSN (Print)10643745

Keywords

  • Epigenetics
  • High-resolution mass spectrometry
  • Histone posttranslational modifications
  • SILAC
  • SILAC spike-in
  • Ultrahigh-pressure liquid chromatography

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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