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.
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.
Zhang
Kangling
K
Department of Chemistry, School of Pharmacology, University of the Pacific, Stockton, California 95211, USA.
Tang
Hui
H
Huang
Lan
L
Blankenship
James W
JW
Jones
Patrick R
PR
Xiang
Fan
F
Yau
Peter M
PM
Burlingame
Alma L
AL
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D006657:Histones; D008780:Methyltransferases; D000123:Acetyltransferases; D014357:Trypsin; D008239:Lysine