22 4 2010 4 PROGRESS IN CHEMISTRY Vol. 22 No. 4 Apr. 2010 * 1 1 1 1 2 1. 300071 2. 116023 bottom-up top-down CID ECD ETD O629. 7 O657. 6 A 1005-281X 2010 04-0713-07 Development of Identification for Post-Translational Modifications in Histones by Mass Spectrometry Based Proteomics Chen Ying 1 Zhang Kai 1 He Xiwen 1 Zhang Yukui 1 2 1. Department of Chemistry Nankai University Tianjin 300071 China 2. National Chromatographic Research and Analysis Center Dalian Institute of Chemical Physics Chinese Academy of Sciences Dalian 116023 China Abstract In eukaryotic cells chromosomal DNA is packaged into a compact structure chromatin with the help of four core histones. The fundamental repeating unit of chromatin is the nucleosome which contains an octamer of core histones around 147 base pairs of DNA are wrapped. Post-translational modifications PTMs in histones are generally considered to be a major group of epigenetic marks. Accordingly because PTMs alter the properties of histones hence affect chromatin structure and function and regulate the gene expression. Therefore identification of histone PTMs is the key to discover histone codes. At present mass spectrometry has become a powerful analysis tool. In this paper we introduced the novel development of the strategies based on mass spectrometry bottom-up and top-down in mapping histone PTMs fragment dissociation technologies such as CID ECD and ETD. We also reported the application of mass spectrometry in addressing histone PTMs sites identifying histone variants and quantifying differential expression of these PTMs in cells. Key words histone post-translational modifications PTMs mass spectrometry high prerormance liquid chromatography HPLC 2009 5 2009 8 * No. 90919008 20975053 U0932001 973 No. 2007CB914100 NN-CAS-2008-8 Corresponding author e-mail Zhangkai730@ yahoo. com. cn
714 22 Contents 1 The strategies and methods of identification for post-translational modifications in histones by mass spectrometry 1. 1 The strategy for mass spectrometry based histone analysis 1. 2 MS fragmentation 2 The application of mass spectrometry for posttranslational modifications in histones 2. 1 Identification and characterization of posttranslational modifications in histones 2. 2 Identification and characterization of histone variants 2. 3 Quantification based mass spectrometry for posttranslational modifications in histones H2A H2B 1 3 a 1 H3 H4 DNA b N H1 Fig. 1 Chromatin organization and tails of histone 3. a 1 4 1a DNA General chromatin organization. b The N-terminal human DNA H3 is shown in single-letter amino-acide code 1 RNA 7 9 5 6 N 10 N N post-translational modifications PTMs 1b 8 60 2 1 ADP 2 Allis 1 DNA 1. 1 bottom-up top-down 2 Edman 1. 1. 1 Bottom-up Edman
4 715 2 bottom-up top-down 14 Fig. 2 Dissecting the primary structure of proteins by mass spectrometry. The bottom-up approach top and the topdown approach bottom 14 LC / MS / MS Zhang 11 2002 14 bottom-up H3 H4 1. 2 Garcia 12 H1 5 2 2 Rose 13 CID / CAD ECD H1t C ETD S140 S162 S177 1. 2. 1 CID S140 CID T155 S162 bottom-up N 2 b- N y- C b-/ y- 19 Kim 20 HPLC / MS / MS A TSA TSA HeLa 14 Boyne 15 HeLa H2A H2A Jiang 16 Gcn5 8 4 H2B H3 Gcn5Δ H4 H2A Parks 17 LC LTQ-FT Ouvry-Patat 18 H2A-Ⅳ IEF-FFE - K5 N top-down H4 CID-MS 1. 1. 2 Top-down H4K5 8 12 16 CID 2 3 21 CID
716 22 22 QLT-obitrap 1. 2. 2 ECD NSI McLafferty FT-ICR FTMS H4 N ICR 1. 14s Cα N 2 555Da c- z- c- N z- C 23 ECD Thomas 24 ECD HeLa H3. 1K4 2. 1 5% K9 K14 H4R3 29 K23 Boyne MS / MS H3K79 15 30 H2A 31 H2A. Z H2A. F / Z α-n 10% H3K56 Siuti 25 ECD H4K91 H4K59 H2B Jiang 16 DNA ECD 32 36 Mollah 37 H3 H2A C K K125 K127 K129 Snijders 38 1. 2. 3 ETD ETD Cα-N c- z- Garcia 39 IMAC H1 6 21 Coon 26 19 Garcia ETD 40 c- z- H3 3 HeLa H3. 1 H3. 2 H3. 3 S10 S28 T3 H3. 1 H2A. Z N H3. 3 S31 C 15 40 Phanstiel 27 HPLC ETD 12 H4 74 IMAC McAlister 28 QLT-obitrap 2 H1 N α - H1 LC / MS / MS ETD
4 717 H1S4 H1S5 13 H1 H1t S140 H1 S162 S177 PTMap DNA 41 42 LC / MS / MS H1 PTMs H1 3 T18 S172 S187 Rose H1. 4 14 3 43 44 3 H3 H4 H2B Ac Me Me2 buty 43 prop Fig. 3 Identified modification sites within histones H3 H4 and H2B from yeast. The known modifications include Ac acetyl Me1 methyl Me2 dimethyl buty butyryl prop propionyl. Unknown modifications are indicated by the sizes of the mass shifts in Daltons 43 15 2. 2 25 MS H2B H2A H3 H2A macroh2a H2A. Z H2A. X 45 47 H2B HTB1 HTB2 HTB4 HTB9 top-down HTB11 48 H2A H2B ECD H3 H2B 7 H2A 14 15 49 51 H2B H2A H1 6 52 H1 53 Garcia H3 H4 bottom-up 54 Middle Down ESI / FTMS / H2A H2B ECD 10 H3
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