title: Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila.
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.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Garcia
authors.forename: Swati
authors.initials: S
authors.lastname: Joshi
authors.forename: C Eric
authors.initials: CE
authors.lastname: Thomas
authors.forename: Raghu K
authors.initials: RK
authors.lastname: Chitta
authors.forename: Robert L
authors.initials: RL
authors.lastname: Diaz
authors.forename: Scott A
authors.initials: SA
authors.lastname: Busby
authors.forename: Philip C
authors.initials: PC
authors.lastname: Andrews
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogorzalek Loo
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Craig A
authors.initials: CA
authors.lastname: Mizzen
authors.forename: C David
authors.initials: CD
authors.lastname: Allis
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D005786:Gene Expression Regulation; D006657:Histones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015800:Protozoan Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013217:Starvation; D016808:Tetrahymena thermophila / Q000254:growth & development / Q000378:metabolism*
chemical_list: D006657:Histones; D010750:Phosphoproteins; D020033:Protein Isoforms; D015800:Protozoan Proteins