Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila

被引:55
作者
Garcia, Benjamin A.
Joshi, Swati
Thomas, C. Eric
Chitta, Raghu K.
Diaz, Robert L.
Busby, Scott A.
Andrews, Philip C.
Loo, Rachel R. Ogorzalek
Shabanowitz, Jeffrey
Kelleher, Neil L.
Mizzen, Craig A.
Allis, C. David
Hunt, Donald F. [1 ]
机构
[1] Univ Virginia, Dept Pathol, Charlottesville, VA 22904 USA
[2] Univ Virginia, Dept Chem, Charlottesville, VA 22904 USA
[3] Rockefeller Univ, Lab Chromatin Biol, New York, NY 10021 USA
[4] Univ Illinois, Dept Chem, Urbana, IL 61801 USA
[5] Univ Illinois, Dept Cell & Dev Biol, Urbana, IL 61801 USA
[6] Univ Calif Los Angeles, Dept Chem & Biochem, Los Angeles, CA 90095 USA
[7] Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48104 USA
关键词
D O I
10.1074/mcp.M600086-MCP200
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1593 / 1609
页数:17
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