title: Global histone profiling by LC-FTMS after inhibition and knockdown of deacetylases in human cells.
abstract: Global histone modifications and their putative relevance to short and long term cellular programming have drawn substantial interest in the study of chromatin. Here we describe the use of reverse-phase liquid chromatography coupled to Linear Ion Trap-Fourier Transform Mass Spectrometry (RPLC-LTQ-FTMS) to quickly profile post-translationally modified isoforms and variants for core histone proteins from as few as 5x10(4) cells at isotopic resolution. Such LC-MS profiling greatly facilitated the detection of histones from HeLa S3 or 293T cells experiencing shRNA- or siRNA-knockdown of histone deacetylase (HDAC) 1, 2, 3 or 1 and 2 together. In no case was significant global histone hyperacetylation relative to control cells observed, suggesting possible compensation of deacetylation activity by partially redundant enzymes in the 18-member HDAC family. This contrasts sharply with yeast where genetic deletion of HDAC rpd3 causes massive hyperacetylation. Treatment of cells with TSA and class I selective HDAC inhibitors had similar ability to induce global histone hyperactylation, though to different extents in HeLa S3 vs. 293T cells.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: Mingxi
authors.initials: M
authors.lastname: Li
authors.forename: Lihua
authors.initials: L
authors.lastname: Jiang
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D000107:Acetylation / Q000187:drug effects; D002460:Cell Line; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D015870:Gene Expression / Q000187:drug effects; D055785:Gene Knockdown Techniques; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D020033:Protein Isoforms / Q000737:chemistry
chemical_list: D056572:Histone Deacetylase Inhibitors; D006657:Histones; D020033:Protein Isoforms; D006655:Histone Deacetylases