title: Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry.
abstract: Histone post-translational modifications (PTMs) are important regulators of chromatin structure and gene expression. Quantitative analysis of histone PTMs by mass spectrometry remains extremely challenging due to the complex and combinatorial nature of histone PTMs. The most commonly used mass spectrometry-based method for high-throughput histone PTM analysis is data-dependent acquisition (DDA). However, stochastic precursor selection and dependence on MS1 ions for quantification impede comprehensive interrogation of histone PTM states using DDA methods. To overcome these limitations, we utilized a data-independent acquisition (DIA) workflow that provides superior run-to-run consistency and postacquisition flexibility in comparison to DDA methods. In addition, we developed a novel DIA-based methodology to quantify isobaric, co-eluting histone peptides that lack unique MS2 transitions. Our method enabled deconvolution and quantification of histone PTMs that are otherwise refractory to quantitation, including the heavily acetylated tail of histone H4. Using this workflow, we investigated the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) on the global histone PTM state of human breast cancer MCF7 cells. A total of 62 unique histone PTMs were quantified, revealing novel SAHA-induced changes in acetylation and methylation of histones H3 and H4.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Krautkramer
authors.affiliation: §BiognoSYS AG, Wagistrasse 25, CH-8952 Schlieren, Switzerland.
authors.forename: Lukas
authors.initials: L
authors.lastname: Reiter
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
authors.forename: James A
authors.initials: JA
authors.lastname: Dowell
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002853:Chromatography, Liquid; D042421:Histone Code / Q000187:drug effects*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D061986:MCF-7 Cells; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation / Q000187:drug effects; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D000077337:Vorinostat
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D010455:Peptides; D000077337:Vorinostat
keywords: DIA; Histone PTM; SAHA; acetylation; data-independent acquisition; epigenetics; mass spectrometry; methylation; proteomics