title: Quantitative proteomic analysis of post-translational modifications of human histones.
abstract: Histone proteins are subject to a range of post-transcriptional modifications in living cells. The combinatorial nature of these modifications constitutes the "histone code" that dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, we used LC-MSMS technology and Virtual Expert Mass Spectrometrist software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational modifications is a viable approach for functional analysis of candidate drugs, such as HDAC inhibitors.
authors.affiliation: Danish Technological Institute, Holbergsvej 10, DK-6000 Kolding, Denmark.
authors.forename: Hans Christian
authors.initials: HC
authors.lastname: Beck
authors.forename: Eva C
authors.initials: EC
authors.lastname: Nielsen
authors.forename: Rune
authors.initials: R
authors.lastname: Matthiesen
authors.forename: Lars H
authors.initials: LH
authors.lastname: Jensen
authors.forename: Maxwell
authors.initials: M
authors.lastname: Sehested
authors.forename: Paul
authors.initials: P
authors.lastname: Finn
authors.forename: Morten
authors.initials: M
authors.lastname: Grauslund
authors.forename: Anne Maria
authors.initials: AM
authors.lastname: Hansen
authors.forename: Ole Nørregaard
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000107:Acetylation / Q000187:drug effects; D000970:Antineoplastic Agents / Q000494:pharmacology; D018288:Carcinoma, Small Cell; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation / Q000187:drug effects; D008958:Models, Molecular; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D013449:Sulfonamides; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; D013449:Sulfonamides; C487081:belinostat