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An experimental design to extract more information from MS-based histone studies

ABSTRACT: The combinatorial complexity of histone samples is manifold higher than what is usually encountered in proteomics. Consequently, a considerably bigger part of the acquired MSMS spectra remains unannotated to date. Adapted search parameters can dig deeper into the dark histone ion space, but the lack of false discovery rate (FDR) control and the high level of ambiguity when searching combinatorial PTMs makes it very hard to assess whether the newly assigned ions are informative. Therefore, we use an easily adoptable time-lapse enzymatic deacetylation (HDAC1) of a commercial histone extract as a quantify-first strategy that allows isolating ion populations of interest, when studying e.g. acetylation on histones, that currently remain in the dark. By adapting search parameters to study potential issues in sample preparation, data acquisition and data analysis, we stepwise managed to double the portion of annotated precursors of interest from 10.5% to 21.6%. This strategy is intended to make up for the lack of validated FDR control and has led to several adaptations of our current workflow that will reduce the portion of the dark histone ion space in the future.

INSTRUMENT(S): SYNAPT G2-Si

ORGANISM(S): Bos Taurus (bovine)

TISSUE(S): Thymus

SUBMITTER: Maarten Dhaenens

LAB HEAD: Maarten Dhaenens

PROVIDER: PXD009910 | Pride | 2022-10-04

REPOSITORIES: Pride

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An experimental design to extract more information from MS-based histone studies.

De Clerck Laura L Willems Sander S Daled Simon S Van Puyvelde Bart B Verhelst Sigrid S Corveleyn Laura L Deforce Dieter D Dhaenens Maarten M

Molecular omics 20211206 6

Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into an alphabet that has more letters than there are amino acids, together making up the "histone code". The resulting combinatorial complexity is manifold higher than what is usually encountered in proteomics. Consequently, a considerably bigger part of the acquire ...[more]

PMID: 34522942

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Project description:Esophageal cancers (ECs) are highly aggressive tumors with poor prognosis and few treatment options. This study investigated the possibility of treating esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells by inhibitors of broad and specific histone deacetylases (HDACi; SAHA, MS-275, FK228) and/or of DNMT (Azacytidine, AZA). Drug targets (HDAC1,2,3 and DNMT1) were present in non-neoplastic (HET-1A), ESCC (OE21) and EAC (OE33) cell lines. All cell lines responded to HDACi by reduced HDAC activity and increased histone acetylation as well as to AZA by up-regulation of p21. Expression of drug targets remained largely unaffected by HDACi and AZA treatment. Importantly, cell viability, apoptosis, cell cycle dynamics and DNA damage were only affected by HDACi and/or AZA in ESCC and EAC, but not the non-neoplastic cells. This was specifically seen for the combination of MS-275 and AZA, leading to enhanced cancer cell selectivity and drug efficiency. By transcriptome analyses of MS-275, AZA and MS-275/AZA treated cells, known (e.g. p21) as well as novel regulated genes significantly associated with the cellular effects post HDACi and/or AZA treatment in ESCC and EAC cells were identified. Finally, human EC tissue specimens frequently expressed the actionable drug targets HDAC1/2/3 and DNMT1. In summary, a combined HDACi (MS-275)/AZA treatment is cancer cell selective and efficient in vitro. Since the majority of ECs express the drug targets in situ, this paves the way for further investigations of HDACi/AZA treatment in esophageal cancer cells and their translation into a clinico-pathological setting. To elucidate the transcriptome response to HDAC inhibitors of normal esophageal cells and esophageal tumor cells, total RNA was isolated from non-neoplastic esophageal epithelial cells (Het1A cells) a well as from two esophageal tumor cell lines (OE21 and OE33), respectively. Cells were treated with either MS-275, Azacytidine (AZA) or in combination of both. DMSO treatment was used as control in each case. Total RNA was isolated from cells 24 h after treatment and experiments were performed in biological triplicates.

Dataset Information

An experimental design to extract more information from MS-based histone studies

Publications

An experimental design to extract more information from MS-based histone studies.

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