Site-specific quantification of lysine acetylation in the N-terminal tail of histone H4 using a double-labelling, targeted UHPLC MS/MS approach.

Annalisa D’Urzo,Vincenza Andrisano,Rainer Bischoff,Alexander P Boichenko,Frank Dekker,Thea Van Den Bosch,Jos Hermans

doi:10.1007/s00216-016-9431-1

Annalisa D’Urzo, Vincenza Andrisano + Show 5 more

Open Access

https://doi.org/10.1007/s00216-016-9431-1

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Journal: Analusis	Publication Date: Mar 11, 2016
Citations: 5	License type: CC BY 4.0

Affiliation: University of Bologna, University of Groningen

Abstract

We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R2 ≥ 0.94), precision (RSD ≤ 10 %), and accuracy (≤27 %) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)–K8 and K5–K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9431-1) contains supplementary material, which is available to authorized users.

Highlights

In eukaryotes, the basic repeating unit of chromatin is composed of 147 bp of DNA wrapped around an octamer of two molecular groups of four histone core proteins: H2A, H2B, H3, and H4
Histone acetylation has been shown to be crucial in hippocampal long-term potentiation (LTP) and memory formation in mice and both aging and Alzheimer’s disease (AD) pathology are associated with a loss of acetylation at the N-terminal tail of histone H4 [1, 2]
Almost all multiple reaction monitoring (MRM) LC-Mass spectrometry (MS)/MS chromatograms for different transitions showed double peaks as shown in Fig. S4

Summary

Introduction

The basic repeating unit of chromatin is composed of 147 bp of DNA wrapped around an octamer of two molecular groups of four histone core proteins: H2A, H2B, H3, and H4. The core histones (∼11–17 kDa) are basic proteins with a globular domain and a flexible N-terminal extension protruding from DNA, referred to as the N-terminal tails. Acetylation of K5, K8, K12, and K16 in the N-terminal tail of histone H4 has been described to play an important role in the epigenetic regulation of cellular events with relevance for diseases like cancer and Alzheimer’s disease (AD). Quantification of histone acetylation on individual lysine residues is of crucial importance to understanding their role in cell biology and disease mechanisms. To this, quantifying histone acetylation will assist in understanding the

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