Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Sara El Kennani,Myriam Ferro,Olga Permiakova,Delphine Pflieger,Côme Ialy-Radio,Anne-Marie Hesse,Annie Adrait,Jérôme Govin,Julie Cocquet,Virginie Brun

doi:10.1186/s13072-017-0172-y

Abstract

BackgroundHistones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Classically, the study of histone variants has largely relied on antibody-based assays. However, antibodies have a limited efficiency to discriminate between highly similar histone variants.ResultsIn this study, we established a mass spectrometry-based analysis to address this challenge. We developed a targeted proteomics method, using selected reaction monitoring or parallel reaction monitoring, to quantify a maximum number of histone variants in a single multiplexed assay, even when histones are present in a crude extract. This strategy was developed on H2A and H2B variants, using 55 peptides corresponding to 25 different histone sequences, among which a few differ by a single amino acid. The methodology was then applied to mouse testis extracts in which almost all histone variants are expressed. It confirmed the abundance profiles of several testis-specific histones during successive stages of spermatogenesis and the existence of predicted H2A.L.1 isoforms. This methodology was also used to explore the over-expression pattern of H2A.L.1 isoforms in a mouse model of male infertility.ConclusionsOur results demonstrate that targeted proteomics is a powerful method to quantify highly similar histone variants and isoforms. The developed method can be easily transposed to the study of human histone variants, whose abundance can be deregulated in various diseases.

Highlights

Histones organize DNA into chromatin through a variety of processes
Theoretical histone peptides relevant for a targeted proteomic analysis The difficulty to perform a functional analysis of histone variants with traditional biochemical approaches comes from their strong sequence homology that exceeds 90% for many of them (Fig. 2)
We hypothesized that targeted proteomics could represent an interesting alternative to the use of antibodies in order to obtain specific detection and precise quantification of histone variants

Summary

Introduction

Histones organize DNA into chromatin through a variety of processes. Among them, a vast diversity of histone variants can be incorporated into chromatin and finely modulate its organization and functionality. Many of them are commercially available and widely utilized by research groups for the quantification and visualization of histones by classical biochemical approaches, such as western blots, immunofluorescence, and immunoprecipitation. These bio-reagents have notably the advantage of being highly sensitive when combined with secondary detection methodologies. They allowed monitoring the abundance of histone variants in several cellular or pathological contexts [9,10,11,12,13,14]

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