Quantitative Mass Spectrometry of Histones H3.2 and H3.3 in Suz12-deficient Mouse Embryonic Stem Cells Reveals Distinct, Dynamic Post-translational Modifications at Lys-27 and Lys-36
Quantitative Mass Spectrometry of Histones H3.2 and H3.3 in Suz12-deficient Mouse Embryonic Stem Cells Reveals Distinct, Dynamic Post-translational Modifications at Lys-27 and Lys-36
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DOI：
10.1074/mcp.m900489-mcp200
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发表时间：
2010-05-01
影响因子：
7
通讯作者：
Jensen, Ole N.
中科院分区：
文献类型：
--
作者：
Jung, Hye Ryung;Pasini, Diego;Jensen, Ole N.
SUZ 12是多梳抑制复合物2（PRC 2）的核心成分，是小鼠胚胎干细胞（ESC）分化所必需的。PRC 2通过H3 Lys-27的甲基化与转录抑制相关。我们应用定量质谱研究Suz 12缺陷对小鼠胚胎干细胞H3.2和H3.3的影响。使用高质量准确度MS结合CID或电子转移解离（ETD）串联质谱，我们从H3.2和H3.3中鉴定了总共81个独特的修饰肽，并在22个不同位置分配了46个修饰，包括不同的共存修饰。在某些情况下，高质量准确度LTQ-Orbitrap MS/MS允许精确定位近同量异位共存的PTM，例如单个肽内的三甲基化和乙酰化。ETD MS/MS促进磷酸化组蛋白肽的测序和注释。ETD和CID MS/MS的组合使用增加了鉴定的修饰肽的总数。在细胞培养物和LC-MS/MS中使用氨基酸稳定同位素标记的来自野生型和Suz 12缺陷型ESC的组蛋白的比较定量分析揭示了H3 K27 me 2和H3 K27 me 3的显著减少和H3 K27 ac的增加，从而揭示了H3 K27处的拮抗性甲基/乙酰基开关。H3 K27甲基化的减少和H3 K27乙酰化的增加伴随着H3 K36乙酰化和甲基化。未修饰和修饰的组蛋白肽（氨基酸27-40）的总体同种型百分比的估计显示了不同的共存组蛋白标记的相对分布。我们的研究揭示了基于抗体的蛋白质印迹方法检测共存的蛋白质修饰的局限性，并证明了定量串联质谱的实用性，用于详细分析蛋白质中共存的翻译后修饰的动态。Molecular & Cellular Proteomics 9：838-850，2010.
SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects of Suz12 deficiency on H3.2 and H3.3 from mouse ESCs. Using high mass accuracy MS combined with CID or electron transfer dissociation (ETD) tandem mass spectrometry, we identified a total of 81 unique modified peptides from H3.2 and H3.3 and assigned 46 modifications at 22 different positions, including distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides. The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone peptides (amino acids 27-40) showed the relative distribution of distinct coexisting histone marks. Our study revealed limitations of antibody-based Western blotting methods for detection of coexisting protein modifications and demonstrated the utility of quantitative tandem mass spectrometry for detailed analysis of the dynamics of coexisting post-translational modifications in proteins. Molecular & Cellular Proteomics 9:838-850, 2010.