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.
Department of Chemistry, School of Pharmacology, University of the Pacific, Stockton, California 95211, USA.
Kangling
K
Zhang
Hui
H
Tang
Lan
L
Huang
James W
JW
Blankenship
Patrick R
PR
Jones
Fan
F
Xiang
Peter M
PM
Yau
Alma L
AL
Burlingame
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