Characterization of phosphorylation sites on histone H1 isoforms by tandem mass spectrometry

J Proteome Res. Nov-Dec 2004;3(6):1219-27. doi: 10.1021/pr0498887.


Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylation
  • Amino Acid Sequence
  • Binding Sites
  • Chromatography, High Pressure Liquid
  • HeLa Cells
  • Histones / chemistry*
  • Histones / metabolism
  • Humans
  • Mass Spectrometry / methods*
  • Methylation
  • Phosphorylation
  • Protein Isoforms / chemistry
  • Protein Isoforms / metabolism
  • Protein Processing, Post-Translational*


  • Histones
  • Protein Isoforms