A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

Abstract

The core histones, H2A, H2B, H3 and H4, undergo post-translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono-, di- and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high-performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom-up liquid chromatography-mass spectrometric analysis. The deuteroacetylation of unmodified or mono-methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross-talk’ by correlating different PTMs on the same histone tail.

The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3

“…To accurately determine site specific lysine acetylation, one can treat samples with deuterated d 6 -acetic anhydride, which converts all unmodified lysines to a "heavy," deuteroacetylated form (Figs. 1C and 1D) (39,40,50,51). The amount of acetylation catalyzed by a HAT enzyme on a specific lysine is calculated using the ratio of "light" (enzymatic) and "heavy" (chemical) acetylation signal detected at that lysine position (Fig.…”

SETD2 reduction adversely affects the development of mouse early embryos

“…1 Multiple important epigenetic changes involving global DNA demethylation, chromatin remodeling, genome spatial reorganization, and substantial transcriptional regulation, initiate to ensure the correct course of embryo development. 4 Histone code, generated from these PTMs, is implicated in transcription, replication, repair, alternative splicing, and other chromatin-related cellular processes. 3 Histone tail domains are modified by substantial posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation, ubiquitinylation, and others.…”

“…Histones are important chromatin proteins through which DNA is structured to manage dynamics of chromosomes . Histone tail domains are modified by substantial posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation, ubiquitinylation, and others . Histone code, generated from these PTMs, is implicated in transcription, replication, repair, alternative splicing, and other chromatin‐related cellular processes …”

“…3 Histone tail domains are modified by substantial posttranslational modifications (PTMs) including phosphorylation, acetylation, methylation, ubiquitinylation, and others. 4 Histone code, generated from these PTMs, is implicated in transcription, replication, repair, alternative splicing, and other chromatin-related cellular processes. 3 SET domain-containing protein 2 (SETD2) is the histone methyltransferase that regulates trimethylation of histone H3 at lysine 36 (H3K36me3), an epigenetic mark commonly associated with gene transcription in mammalian cells.…”

A new approach combining LC-MS and multivariate statistical analysis for revealing changes in histone modification levels

“…Recent studies relying on mass spectrometry explored the histone acetylation from a quantitative and a dynamic point of view at the peptide level. 30,31 In order to remove the difficulties associated with the digestion step and with the peptide characterisation and quantitation, we put our efforts on establishing a new analytical approach for the differential analysis of histone PTMs at the intact protein level by combining the use of UPLC-QTOF-MS and multivariate statistical analysis (MVA). Supervised and unsupervised statistical methods have been widely used in different engineering or scientific disciplines for several years, and more recently in -omic sciences.…”