TY - JOUR
T1 - 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
AU - Zhang, Kangling
AU - Tang, Hui
AU - Huang, Lan
AU - Blankenship, James W.
AU - Jones, Patrick R.
AU - Xiang, Fan
AU - Yau, Peter M.
AU - Burlingame, Alma L.
N1 - Funding Information:
Mass spectral data were provided by the Mass Spectrometry Facility of UC–Davis and by the UCSF Mass Spectrometry Facility supported by the Biomedical Research Technology Program of the National Center for Research Resources, NIH NCRR BRTP RR01614 to A.L.B. The support of Professor A.L. Burlingame is gratefully acknowledged.
PY - 2002/7/15
Y1 - 2002/7/15
N2 - 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.
AB - 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.
KW - Histone acetylation
KW - Histone methylation
KW - MALDI-PSD
KW - MALDI-TOF
KW - Nano-ESI tandem mass
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U2 - 10.1006/abio.2002.5719
DO - 10.1006/abio.2002.5719
M3 - Article
C2 - 12123664
AN - SCOPUS:0037099664
SN - 0003-2697
VL - 306
SP - 259
EP - 269
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -