Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples

Camilla Restellini, Alessandro Cuomo, Michela Lupia, Marco Giordano, Tiziana Bonaldi, Roberta Noberini

Research output: Contribution to journalArticlepeer-review


Purpose: Profiling histone posttranslational modifications (PTMs) in clinical samples holds great potential for the identification of epigenetic biomarkers and the discovery of novel epigenetic targets. MS-based approaches to analyze histone PTMs in clinical samples usually rely on SDS-PAGE separation following histone enrichment in order to eliminate detergents and further isolate histones. However, this limits the digestions options and hence the modification coverage. Experimental design and results: The aim of this study is the implementation of a procedure involving acetone protein precipitation followed by histone enrichment through a C18 StageTip column to obtain histone preparations suitable for various in-solution digestion protocols. Among them, the Arg-C digestion, which allows profiling histone H4 modifications, and the Prop-PIC method, which improves the detection of short and hydrophilic peptides, are tested. This approach is validated on different types of samples, including formalin-fixed paraffin-embedded pathology tissues, and employed to profile histone H4 modifications in cancer samples and normal tissues, identifying previously reported differences, as well as novel ones. Conclusions and clinical relevance: This protocol widens the number of applications available in the toolbox of clinical epigenomics, allowing the investigation of a larger spectrum of histone marks in patient samples.

Original languageEnglish
Article number1700166
JournalProteomics - Clinical Applications
Issue number1
Publication statusPublished - Jan 2019


  • epigenetic mark
  • histone posttranslational modifications
  • mass spectrometry
  • proteomics
  • reversed-phase chromatography

ASJC Scopus subject areas

  • Clinical Biochemistry


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