Comprehensive phosphoprotein analysis of linker histone H1 from tetrahymena thermophila

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, Donald F. Hunt

Research output: Contribution to journalArticle

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, INIAC, and tandem mass spectrometry.

Original languageEnglish (US)
Pages (from-to)1593-1609
Number of pages17
JournalMolecular and Cellular Proteomics
Volume5
Issue number9
DOIs
StatePublished - Sep 1 2006

Fingerprint

Tetrahymena thermophila
Phosphorylation
Phosphoproteins
Histones
Mass spectrometry
Tetrahymena
Mass Spectrometry
Acetylation
Phosphopeptides
Gene Expression Regulation
Tandem Mass Spectrometry
Starvation
Chromatography
Gene expression
DNA Repair
Labeling
Cations
Digestion
Protein Isoforms
Repair

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Garcia, B. A., Joshi, S., Thomas, C. E., Chitta, R. K., Diaz, R. L., Busby, S. A., ... Hunt, D. F. (2006). Comprehensive phosphoprotein analysis of linker histone H1 from tetrahymena thermophila. Molecular and Cellular Proteomics, 5(9), 1593-1609. https://doi.org/10.1074/mcp.M600086-MCP200

Comprehensive phosphoprotein analysis of linker histone H1 from tetrahymena thermophila. / Garcia, Benjamin A.; Joshi, Swati; Thomas, C. Eric; Chitta, Raghu K.; Diaz, Robert L.; Busby, Scott A.; Andrews, Philip C.; Ogorzalek Loo, Rachel R.; Shabanowitz, Jeffrey; Kelleher, Neil L.; Mizzen, Craig A.; Allis, C. David; Hunt, Donald F.

In: Molecular and Cellular Proteomics, Vol. 5, No. 9, 01.09.2006, p. 1593-1609.

Research output: Contribution to journalArticle

Garcia, BA, Joshi, S, Thomas, CE, Chitta, RK, Diaz, RL, Busby, SA, Andrews, PC, Ogorzalek Loo, RR, Shabanowitz, J, Kelleher, NL, Mizzen, CA, Allis, CD & Hunt, DF 2006, 'Comprehensive phosphoprotein analysis of linker histone H1 from tetrahymena thermophila', Molecular and Cellular Proteomics, vol. 5, no. 9, pp. 1593-1609. https://doi.org/10.1074/mcp.M600086-MCP200
Garcia, Benjamin A. ; Joshi, Swati ; Thomas, C. Eric ; Chitta, Raghu K. ; Diaz, Robert L. ; Busby, Scott A. ; Andrews, Philip C. ; Ogorzalek Loo, Rachel R. ; Shabanowitz, Jeffrey ; Kelleher, Neil L. ; Mizzen, Craig A. ; Allis, C. David ; Hunt, Donald F. / Comprehensive phosphoprotein analysis of linker histone H1 from tetrahymena thermophila. In: Molecular and Cellular Proteomics. 2006 ; Vol. 5, No. 9. pp. 1593-1609.
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