Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila.
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.
Garcia
Benjamin A
BA
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Joshi
Swati
S
Thomas
C Eric
CE
Chitta
Raghu K
RK
Diaz
Robert L
RL
Busby
Scott A
SA
Andrews
Philip C
PC
Ogorzalek Loo
Rachel R
RR
Shabanowitz
Jeffrey
J
Kelleher
Neil L
NL
Mizzen
Craig A
CA
Allis
C David
CD
Hunt
Donald F
DF
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*
D006657:Histones; D010750:Phosphoproteins; D020033:Protein Isoforms; D015800:Protozoan Proteins