Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes—a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC–MS

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.

Natural isotope correction improves analysis of protein modification dynamics

“…As acetyl-CoA is also the substrate for histone acetylations, natural occurring 12 C-acetylation of lysines are replaced with 13 C-labelled acetyl-groups causing a characteristic mass shift of the peptide in high-resolution MS spectra. The time-dependent replacement of 12 C-acetylation can be used to determine histone acetylation dynamics [ 8 , 9 , 13 , 14 ].

Fig.

…”

“…Molecules with the same composition of isotopes, so-called isotopomers, are chemically identical. Their intact precursor ions are isobaric and therefore indistinguishable by MS analysis, [ 9 ] with deuterium labelling leading to different chromatographic behavior in reversed-phase chromatography due to the higher polarity of deuterium. An additional fragmentation by tandem mass spectrometry (MS/MS or MS2) using, e.g., collision induced dissociation (CID), allows to localize the isotopes based on their presence in the individual fragments [ 9 ].…”

“…Their intact precursor ions are isobaric and therefore indistinguishable by MS analysis, [ 9 ] with deuterium labelling leading to different chromatographic behavior in reversed-phase chromatography due to the higher polarity of deuterium. An additional fragmentation by tandem mass spectrometry (MS/MS or MS2) using, e.g., collision induced dissociation (CID), allows to localize the isotopes based on their presence in the individual fragments [ 9 ]. The obtained data need to be appropriately processed, in order to correctly calculate metabolite or protein modification dynamics [ 9 ].…”

“…An additional fragmentation by tandem mass spectrometry (MS/MS or MS2) using, e.g., collision induced dissociation (CID), allows to localize the isotopes based on their presence in the individual fragments [ 9 ]. The obtained data need to be appropriately processed, in order to correctly calculate metabolite or protein modification dynamics [ 9 ]. A further complication in this analysis is the necessity to correct the obtained values for natural isotope abundance, called isotop(ologu)e correction or deconvolution.…”