Label-Free Mass Spectrometry-Based Quantification of Linker Histone H1 Variants in Clinical Samples

Giuseppe Diaferia,Paola Scaffidi,Cristina Morales Torres,Giovanni Bertalot,Roberta Noberini,Maria Giovanna Jodice,Stefania Brandini,Giuseppina Bonizzi,Evelyn Oliva Savoia,Tiziana Bonaldi,Maria Capra

doi:10.3390/ijms21197330

Abstract

Epigenetic aberrations have been recognized as important contributors to cancer onset and development, and increasing evidence suggests that linker histone H1 variants may serve as biomarkers useful for patient stratification, as well as play an important role as drivers in cancer. Although traditionally histone H1 levels have been studied using antibody-based methods and RNA expression, these approaches suffer from limitations. Mass spectrometry (MS)-based proteomics represents the ideal tool to accurately quantify relative changes in protein abundance within complex samples. In this study, we used a label-free quantification approach to simultaneously analyze all somatic histone H1 variants in clinical samples and verified its applicability to laser micro-dissected tissue areas containing as low as 1000 cells. We then applied it to breast cancer patient samples, identifying differences in linker histone variants patters in primary triple-negative breast tumors with and without relapse after chemotherapy. This study highlights how label-free quantitation by MS is a valuable option to accurately quantitate histone H1 levels in different types of clinical samples, including very low-abundance patient tissues.

Highlights

In the eukaryotic nucleus, genomic DNA is packed into chromatin, which regulates all nuclear processes involving DNA, including DNA replication, DNA repair and transcription
We show the feasibility of applying a simple label-free Mass spectrometry (MS)-based approach for the quantitative and simultaneous analysis of histone H1 variant levels from clinical samples
This method takes advantage of the MaxLFQ algorithm, which has been extensively used in quantitative global proteomics studies, but whose application to protein isoforms, such as histone H1 variants, has not been investigated so far

Summary

Introduction

Genomic DNA is packed into chromatin, which regulates all nuclear processes involving DNA, including DNA replication, DNA repair and transcription. Linker histone H1 binds the free DNA (~20 bp-long) present between individual nucleosomes, contributing to the formation of higher-order chromatin structures. The nucleosome, linker DNA and linker histone H1 form the chromatosome. Core histones exert their function mainly through a number of reversible post-translational modifications (PTMs) that can be deposed at their N-terminal tails, whose role in regulating gene expression has been extensively studied and elucidated in recent years [1]. Much less is known regarding the role of histone H1 besides its general chromatin condensation function, increasing evidence indicates histone H1 potential to regulate transcription in a locus-specific manner [2,3,4]

Methods

Results

Conclusion