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A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling.

ABSTRACT: Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d₀-acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.

SUBMITTER: Zappacosta F

PROVIDER: S-EPMC8138768 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Precision medicine has been strongly promoted in recent years. It is used in clinical management for classifying diseases at the molecular level and for selecting the most appropriate drugs or treatments to maximize efficacy and minimize adverse effects. In precision medicine, an in-depth molecular understanding of diseases is of great importance. Therefore, in the last few years, much attention has been given to translating data generated at the molecular level into clinically relevant information. However, current developments in this field lack orderly implementation. For example, high-quality chemical research is not well integrated into clinical practice, especially in the early phase, leading to a lack of understanding in the clinic of the chemistry underlying diseases. In recent years, mass spectrometry (MS) has enabled significant innovations and advances in chemical research. As reported, this technique has shown promise in chemical mapping and profiling for answering "what", "where", "how many" and "whose" chemicals underlie the clinical phenotypes, which are assessed by biochemical profiling, MS imaging, molecular targeting and probing, biomarker grading disease classification, etc. These features can potentially enhance the precision of disease diagnosis, monitoring and treatment and thus further transform medicine. For instance, comprehensive MS-based biochemical profiling of ovarian tumors was performed, and the results revealed a number of molecular insights into the pathways and processes that drive ovarian cancer biology and the ways that these pathways are altered in correspondence with clinical phenotypes. Another study demonstrated that quantitative biomarker mapping can be predictive of responses to immunotherapy and of survival in the supposedly homogeneous group of breast cancer patients, allowing for stratification of patients. In this context, our article attempts to provide an overview of MS-based chemical mapping and profiling, and a perspective on their clinical utility to improve the molecular understanding of diseases for advancing precision medicine.

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A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling.

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