Abstract
Histone acetylation is an important, reversible post-translational protein modification and a hallmark of epigenetic regulation. However, little is known about the dynamics of this process, due to the lack of analytical methods that can capture site-specific acetylation and deacetylation reactions. We present a new approach that combines metabolic and chemical labeling (CoMetChem) using uniformly 13C-labeled glucose and stable isotope-labeled acetic anhydride. Thereby, chemically equivalent, fully acetylated histone species are generated, enabling accurate relative quantification of site-specific lysine acetylation dynamics in tryptic peptides using high-resolution mass spectrometry. We show that CoMetChem enables site-specific quantification of the incorporation or loss of lysine acetylation over time, allowing the determination of reaction rates for acetylation and deacetylation. Thus, the CoMetChem methodology provides a comprehensive description of site-specific acetylation dynamics.
Highlights
Living organisms are highly dynamic systems and constantly exposed to external changes and strive to maintain homeostasis
Complete chemical acetylation of the nonacetylated lysine residues at the protein level is essential for the CoMetChem strategy to ensure that only chemically equivalent species arise from the tryptic digestion of a given protein
HDACi treatment did not result in higher acetylation levels for the single-acetylated H4(4-17) species, analysis of site-specific reaction rates revealed that treatment with Mass spectrometry (MS)-275 and suberanilohydroxamic acid (SAHA) resulted in complete inhibition of deacetylation reactions at K16 (d4) compared to the control (Figure S6B) and to decreased acetylation rates at the same site (Figure S6C)
Summary
Living organisms are highly dynamic systems and constantly exposed to external changes and strive to maintain homeostasis. In contrast to deacetylation rates, the CoMetChem approach allows us to determine site-specific acetylation rates for the formation of the double-acetylated peptide This is based on the conversion of the single-acetylated H3(18-26). HDACi treatment did not result in higher acetylation levels for the single-acetylated H4(4-17) species, analysis of site-specific reaction rates revealed that treatment with MS-275 and SAHA resulted in complete inhibition of deacetylation reactions at K16 (d4) compared to the control (Figure S6B) and to decreased acetylation rates at the same site (Figure S6C). Treatment with MS-275 resulted in higher acetylation rates at K23 for the double-acetylated (a3) species compared to the control, while SAHA and the control showed similar acetylation rates Taken together, these results demonstrate that our CoMetChem approach provides a comprehensive description of the dynamics of reversible lysine acetylation by determining the site-specific reaction rates of deacetylation and acetylation. Using CoMetChem, we were able to describe the dynamic effects of HDACs in a site-specific manner using HDAC inhibitors
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