Investigating pathological epigenetic aberrations by epi-proteomics

Abstract

Epigenetics includes a complex set of processes that alter gene activity without modifying the DNA sequence, which ultimately determines how the genetic information common to all the cells of an organism is used to generate different cell types. Dysregulation in the deposition and maintenance of epigenetic features, which include histone posttranslational modifications (PTMs) and histone variants, can result in the inappropriate expression or silencing of genes, often leading to diseased states, including cancer. The investigation of histone PTMs and variants in the context of clinical samples has highlighted their importance as biomarkers for patient stratification and as key players in aberrant epigenetic mechanisms potentially targetable for therapy. Mass spectrometry (MS) has emerged as the most powerful and versatile tool for the comprehensive, unbiased and quantitative analysis of histone proteoforms. In recent years, these approaches-which we refer to as "epi-proteomics"-have demonstrated their usefulness for the investigation of epigenetic mechanisms in pathological conditions, offering a number of advantages compared with the antibody-based methods traditionally used to profile clinical samples. In this review article, we will provide a critical overview of the MS-based approaches that can be employed to study histone PTMs and variants in clinical samples, with a strong focus on the latest advances in this area, such as the analysis of uncommon modifications and the integration of epi-proteomics data into multi-OMICs approaches, as well as the challenges to be addressed to fully exploit the potential of this novel field of research.

Keywords: Cancer; Epigenetics; Histone posttranslational modification; Histone variant; Histone-modifying enzyme; Mass spectrometry; Proteomics.

Fig. 1

Role of histones in the regulation of gene expression. Histone posttranslational modifications (PTMs) and variants contribute to the regulation of the expression of genes, determining changes at the levels of transcripts, proteins and metabolites that can lead to aberrant phenotypes. In turn, proteins and metabolites can influence the levels and the effects of histone PTMs and variants, by affecting the levels of histone-modifying enzymes (HMEs), histone chaperones and readers, and intermediate metabolites

Fig. 2

Epi-proteomics approaches for histone analysis. Scheme summarizing the three main MS-based approaches applicable for histone PTM and variant analysis. aa: amino acid

Fig. 3

Schematic bottom-up workflow for MS-based analysis of histone PTMs and variants. Histones are enriched through specific protocols from different types of clinical samples, separated by SDS-PAGE and in-gel digested. Digested peptides are separated by liquid chromatography and acquired in the mass spectrometer. MS spectra are then used for peptide identification and quantification, and PTM assignment

Fig. 4

MALDI imaging workflow. Tissue sections are mounted on slides appropriate for MALDI imaging, are digested with trypsin (as an optional step) and covered with a MALDI matrix. The tissue slides are scanned by a laser beam, which generates MS spectra for each xy coordinate. MALDI imaging profiles can guide the selection of area of interest to be laser microdissected and analyzed by LC/MS