Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry

Kangling Zhang, Hui Tang, Lan Huang, James W. Blankenship, Patrick R. Jones, Fan Xiang, Peter M. Yau, Alma L. Burlingame

Research output: Contribution to journalArticle

122 Scopus citations

Abstract

A new strategy has been employed for the identification of the covalent modification sites (mainly acetylation and methylation) of histone H3 from chicken erythrocytes using low enzyme/substrate ratios and short digestion times (trypsin used as the protease) with analysis by HPLC separation, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), matrix-assisted laser desorption ionization-post-source decay, and tandem mass spectrometric techniques. High-accuracy MALDI-TOF mass measurements with representative immonium ions (126 for acetylated lysine, 98 for monomethylated lysine, and 84 for di-, tri-, and unmethylated lysine) have been effectively used for differentiating methylated peptides from acetylated peptides. Our results demonstrate that lysines 4, 9, 14, 27, and 36 of the N-terminal of H3 are methylated, while lysines 14, 18, and 23 are acetylated. Surprisingly, a non-N-terminal residue, lysine 79, in the loop region hooking up to the bound DNA, was newly found to be methylated (un-, mono-, and dimethylated isoforms coexist). The reported mass spectrometric method has the advantages of speed, directness, sensitivity, and ease over protein sequencing and Western-blotting methods and holds the promise of an improved method for determining the status of histone modifications in the field of chromosome research.

Original languageEnglish (US)
Pages (from-to)259-269
Number of pages11
JournalAnalytical Biochemistry
Volume306
Issue number2
DOIs
StatePublished - Jul 15 2002
Externally publishedYes

Keywords

  • Histone acetylation
  • Histone methylation
  • MALDI-PSD
  • MALDI-TOF
  • Nano-ESI tandem mass

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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