Analysis of histone modifications from tryptic peptides of deuteroacetylated isoforms

Abstract

The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.

High Energy Collisions on Tandem Time-of-Flight Mass Spectrometers

“…Figure 17 shows the high energy CID MS/MS spectra of the monoacetylated and diacetylated species obtained on a Shimadzu AXIMA TOF 2 mass spectrometer. The observed b -series and y -series ions are consistent with the major isoforms being monoacetylation at K 16 and diacetylation at K 8 and K 16 (48). …”

HDAC 3-selective inhibitor RGFP966 demonstrates anti-inflammatory properties in RAW 264.7 macrophages and mouse precision-cut lung slices by attenuating NF-κB p65 transcriptional activity

“…In order to assess the changes in lysine acetylation on specific residues, histone acetylation of histone H3 and H4 was analyzed using mass spectrometry. To enable quantification, a previously described approach was employed in which the histones were fully acetylated using deuterated acetic acid anhydride ((CD 3 CO) 2 O) as a reagent [31] , [32] , [33] , [34] . Using this method deuterated acetyl groups were installed on non-acetylated lysine residues.…”

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.…”