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Molecular & Cellular Proteomics 5:1593-1609, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Comprehensive Phosphoprotein Analysis of Linker Histone H1 from Tetrahymena thermophila^*,S

Benjamin A. Garcia^,, Swati Joshi^¶, C. Eric Thomas^||, Raghu K. Chitta, Robert L. Diaz^¶, Scott A. Busby, Philip C. Andrews^**, Rachel R. Ogorzalek Loo, Jeffrey Shabanowitz, Neil L. Kelleher^||, Craig A. Mizzen, C. David Allis^¶ and Donald F. Hunt^,¶¶,||||

From the Departments of Chemistry and ^¶¶ Pathology, University of Virginia, Charlottesville, Virginia 22904, ^¶ Laboratory of Chromatin Biology, The Rockefeller University, New York, New York, 10021, Departments of ^|| Chemistry and Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, and ^** Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104

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.

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