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

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

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 199 KB
• Volume: 306
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2002.5719

✦ Synopsis

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-postsource 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.