Proteo-Metabo-Flux – linking Acetyl-CoA and acetylation dynamics for accurate determination of histone acetylation rates

Introduction

Histone acetylation is an important post-translational modification that regulates DNA accessibility and gene translation. The substrate for this modification is the small molecule acetyl-CoA, which plays a central role in carbon metabolism. To fully understand the biological function of this reversible post-translational modification it is not sufficient to determine static acetylation levels but it is necessary to capture individual acetylation and deacetylation reaction rates. We have developed a quantitative proteo-metabo-flux approach and pipeline (Proteo-Metabo-Flux-Quant, PMF-Quant) including the simultaneous extraction of acetyl-CoA and histones from cell culture as well as normalisation and correction algorithms to quantify site-specific histone acetylation rates and to combine them with acetyl-CoA turnover dynamics.

Results

Here, we present our global Proteo-Metabo-Flux-Quant (PMF-Quant) pipeline for the analysis of acetyl-CoA and histone acetylation dynamics. We use metabolic labelling with [U-13]-C glucose followed by a simultaneous extraction of acetyl-CoA and histones [1]. Histones are tchemically acetylated with 13C4,D6-acetic anhydride and digested using trypsine to obtain chemically equivalent peptides [2]. Ordinary differential equations are used to build an integrated model of label incorporation into both acetyl-CoA and histone acetyl groups. We find that 13C from glucose is incorporated into the acetyl- as well as the ribose and adenine moieties of acetyl-CoA over a of 6 hour period, with label incorporation plateauing at approximately 70%. Taking this partial incorporation into account is important when calculating histone acetylation rates, which are otherwise greatly underestimated. As a proof of principle, we treat cells with the Akt inhibitor MK-2206, which inhibits glycolysis. This leads to an apparent change in histone acetylation rates when the correction is omitted, which is shown to be an artefact of upstream effects.

References

[1] van Pijkeren, A. et al, Proteome Coverage after Simultaneous Proteo-Metabolome Liquid–Liquid Extraction. Journal of Proteome Research 2023, 22, 3, 951-966

[2] van Pijkeren, A. et al., Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes—a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC–MS. Analytical Chemistry 2021, 93, 38, 12872-12880