Alternative digestion approaches improve histone modification mapping by mass spectrometry in clinical samples. Issue 1 (6th December 2018) - Restellini, Camilla
Abstract : 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.