Identification of histone Khib substrates regulated by NAT10
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Kai Zhang
PROVIDER: PXD073624 | iProX | Tue Jan 27 00:00:00 GMT 2026
REPOSITORIES: iProX
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Project description:Having established NAT10’s regulatory role in lysine 2-hydroxyisobutyrylation, we aimed to identify the histone Khib sites targetedby NAT10. To this end, we extracted and digested core histones from cells with or without NAT10 depletion, followed by chemical derivatization of amine groups. The resulting peptides were analyzed by data-independent acquisition (DIA) mass spectrometry to detect the NAT10-modulated Khib sites.
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Project description:Histone post-translational modifications (hPTMs) belong to decisive epigenetic marks affecting, among others, cell cycle and protein interactions. hPTMs have been studied by both antibody- and mass spectrometry-based methods for years, however, those analyses are still challenging, mainly due to multiple histone variants and their modification diversity arising from high content of reactive amine groups in the amino acid sequence. Here, we introduce trimethylacetic anhydride (TMA) as a new reagent for efficient histone alkylation, a necessary approach prior to hPTMs analysis using bottom-up proteomics. TMA reagent is applied for derivatization of unmodified amine groups at lysine residues as well as for subsequent alkylation of newly generated amine groups at peptide N-termini after trypsin digestion. Derivatization reaction is facilitated by microwave irradiation, which also reduces incubation time to minute intervals. We demonstrate that histone alkylation with TMA represents a reliable technique with high yield of fully derivatized peptides, hence an effective alternative to conventionally used methods. More than 98 % and 99 % labelling efficiency of TMA was achieved for histone H4 and H3, respectively, ensuring an accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms which is a prerequisite for direct quantification based on signal extracted from MS1 data. For this purpose, software tools widely adopted within proteomics community can be used without a need of additional computational advances. Based on a thorough side-by-side comparison of TMA performance with well-adopted propionylation we comment on the impact of histone derivatization on the hPTMs monitoring in biological samples.
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Project description:The posttranslational modification lysine malonylation is found in many proteins, including histones. However, it remains unclear whether histone malonylation is regulated or functionally relevant. Here, we report that availability of malonyl-co-enzyme A (malonyl-CoA), an endogenous provider of malonyl groups, affects lysine malonylation, and that the deacylase SIRT5 selectively reduces malonylation of histones. To determine if histone malonylation is enzymatically catalyzed, we knocked down each of the 22 lysine acetyltransferases (KATs) to test their malonyltransferase potential. KAT2A knockdown reduced and KAT2A overexpression increased levels of histone malonylation, respectively. By mass spectrometry, H2B_K5 was highly malonylated and significantly regulated by SIRT5 in mouse brain and liver. Acetyl-CoA carboxylase (ACC), the malonyl-CoA producing enzyme, was partly localized in the nucleolus, and histone malonylation increased nucleolar area and ribosomal RNA expression. Levels of global lysine malonylation and ACC expression were higher in older mouse brains than younger mice. These experiments highlight the role of histone malonylation in ribosomal gene expression.
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Project description:Lysine 2-hydroxyisobutyrylation (Khib) is a novel type of histone acylation in eukaryotes, but its prevalence and genomic function in higher plants remain unclear. Using mass spectrometry, we identified histone lysine 2-hydroxyisobutyrylation in Arabidopsis
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Project description:Histone lysine methylation is an important post-translational modifications (PTMs) that plays critical roles in numerous biological processes with abnormal histone lysine methylation having been associated with developmental defects and human diseases. The primary amine of all lysine residues can be mono-, di-, or trimethylated and the extent of methylation at a single site is shown to inspire unique protein function. Moreover, histone lysine methylation can activate or repress transcription depending on which sites are modified and to what degree. Therefore, a comprehensive stoichiometric analysis of histone lysine methylation is necessary to fully elucidate its function. As histones are lysine-rich and highly-hydrophilic proteins (Figure S1), trypsin digestion of histones results in small, hydrophilic peptides which often suffer from substantial losses during sample preparation, and are difficult to detect in conventional reversed phase LC-MS. Additionally, trypsin fails to cleave histone proteins when lysine residues are methylated, resulting in inaccurate estimations of site occupancy and methylation extent. Previously, lysine propionylation labeling has been developed to overcome this drawback and facilitate quantitative analysis of PTMs that frequently occur on the lysine residue of histones. However, propionylation labeling of unmodified and monomethylated lysine residues causes a mismatch in hydrophobicity and charge state between labeled and unlabeled di-/trimethylated peptides. This mismatch forces retention time and signal intensity differences that inspire inaccurate quantitation and miscalculation of site occupancy. In this work, we developed a method that facilitates blocking of free lysine groups and discrimination of native lysine methylation, enables accurate calculation of the histone lysine methylation stoichiometry.
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Project description:Bottom-up proteomics approach has become an important strategy in diverse areas of biological research, and the enzymatic digestion is essential for this technology. Endopeptidase Arg-C catalyzing the hydrolytic cleavage of peptide bonds C-terminal to arginine could be an important protease in bottom-up proteomics. However, it has been seldom applied due to its low specificity and high cost. Reversible chemical derivatization of amine allows a real Arg-C digestion using trypsin. In this paper, a reversible amine derivatization method, citraconylation and decitraconylation were optimized and evaluated for large-scale proteomics studies. Combination of the reversible derivatization and trypsin digestion (termed Arg-C* digestion to distinguish from the conventional Arg-C digestion) resulted in 64.2% more peptide identification (11,925 ± 199 vs 7,262 ± 59) and significantly higher cleavage specificity (95.6% vs 73.6%) than the conventional Arg-C digestion. Comparison of Arg-C* digestion with the widely used trypsin and Lys-C digestion revealed that Arg-C* performed equally or slightly better than Lys-C although not comparable to trypsin. Therefore, the well-established Arg-C* digestion method is a promising approach for proteomics studies and could be used as the prior alternative digestion method to trypsin digestion in order to achieve higher proteome coverage.
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