[Show abstract][Hide abstract] ABSTRACT: Analyses of histone H3 from 10 rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissociation showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addition, a novel rat testes-specific H3 protein was identified with this approach.
Journal of Proteome Research 09/2008; 7(10):4225-36. DOI:10.1021/pr800044q · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Post-translational modifications (PTMs) of histones are intimately involved in chromatin structure and thus have roles in cellular processes through their impact on gene activation or repression. At the forefront in histone PTM analysis are mass spectrometry-based techniques, which have capabilities to produce improved views of processes affected by chromatin remodeling via histone modifications. In this report, we take the first mass spectrometric look at histone variant expression and post-translational modifications from histones isolated from rat brain tissue. Analyses of whole rat brain identified specific histone H2A and H2B gene family members and several H4 and H3 post-translational modification sites by electron capture dissociation (ECD) mass spectrometry. We subsequently compared these results to selected rat brain regions. Major differences in the expression profiles of H2A and H2B gene family members or in the post-translational modifications on histone H4 were not observed from the different brain regions using a Top Down approach. However, “Middle Down” mass spectrometry facilitating improved characterization of the histone H3 tail (1–50 residues), revealed an enrichment of trimethylation on Lys9 from cerebellum tissue compared to H3 extracted from whole brain, cerebral cortex or hypothalamus tissue. We forward this study in honor of Professor Donald F. Hunt, whose pioneering efforts in protein and PTM analyses have spawned new eras and numerous careers, many exemplified in this special issue.
International Journal of Mass Spectrometry 01/2007; 259(1-259):184-196. DOI:10.1016/j.ijms.2006.07.022 · 1.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Linker histone H1 is highly phosphorylated in normal growing Tetrahymena thermophila but becomes noticeably dephosphorylated in response to certain conditions such as prolonged starvation. Because phosphorylation of H1 has been associated with the regulation of gene expression, DNA repair, and other critical processes, we sought to use mass spectrometry-based approaches to obtain an in depth phosphorylation "signature" for this linker histone. Histone H1 from both growing and starved Tetrahymena was analyzed by nanoflow reversed-phase HPLC MS/MS following enzymatic digestions, propionic anhydride derivatization, and phosphopeptide enrichment via IMAC. We confirmed five phosphorylation sites identified previously and detected two novel sites of phosphorylation and two novel minor sites of acetylation. The sequential order of phosphorylation on H1 was deduced by using mass spectrometry to define the modified sites on phosphorylated H1 isoforms separated by cation-exchange chromatography. Relative levels of site-specific phosphorylation on H1 isolated from growing and starved Tetrahymena were obtained using a combination of stable isotopic labeling, IMAC, and tandem mass spectrometry.
[Show abstract][Hide abstract] ABSTRACT: The modification of H3 in asynchronous HeLa cells was profiled using Top Down Mass Spectrometry. A broad distribution of species differing by 14 Da and containing less than 3% unmodified protein was observed for all three variants. Species of up to +168 Da were observed for H3.1, and fragmentation of all species by Electron Capture Dissociation (ECD) revealed approximately 5% methylation of K4 and approximately 50% dimethylation of K9. K14 and K23 were major sites of acetylation. H3.3 was slightly hypermodified with the apex of the distribution shifted by approximately +14 Da compared to H3.1. H3.1 (50% and 15%) from colchicine-treated cells was monophosphorylated and diphosphorylated, respectively, with equivalent modification of S10 and S28.
Journal of Proteome Research 03/2006; 5(2):240-7. DOI:10.1021/pr050266a · 4.25 Impact Factor