Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry
Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry
复制标题
DOI：
10.1021/acs.jproteome.5b00245
复制
发表时间：
2015-08-01
影响因子：
4.4
通讯作者：
Dowell, James A.
中科院分区：
文献类型：
--
作者：
Krautkramer, Kimberly A.;Reiter, Lukas;Dowell, James A.
组蛋白翻译后修饰（PTM）是染色质结构和基因表达的重要调控因子。由于组蛋白PTM的复杂性和组合性，通过质谱法定量分析组蛋白PTM仍然极具挑战性。用于高通量组蛋白PTM分析的最常用的基于质谱的方法是数据依赖采集（DDA）。然而，随机的前体选择和依赖于MS 1离子的定量阻碍了使用DDA方法对组蛋白PTM状态的全面询问。为了克服这些限制，我们使用了数据独立采集（DIA）工作流程，与DDA方法相比，该工作流程提供了卓越的上级运行一致性和采集后灵活性。此外，我们开发了一种新的DIA为基础的方法来量化同量异位，共洗脱组蛋白肽，缺乏独特的MS2转换。我们的方法能够对组蛋白PTM进行去卷积和定量，否则这些PTM难以定量，包括组蛋白H4的高度乙酰化的尾部。使用这个工作流程，我们研究了组蛋白去乙酰化酶抑制剂SAHA（辛二酰苯胺异羟肟酸）对人乳腺癌MCF 7细胞的全局组蛋白PTM状态的影响。共62个独特的组蛋白PTM进行了定量，揭示了新的SAHA诱导的组蛋白H3和H4的乙酰化和甲基化的变化。
Histone post-translational modifications (PTMs) are important regulators of chromatin structure and gene expression. Quantitative analysis of histone PTMs by mass spectrometry remains extremely challenging due to the complex and combinatorial nature of histone PTMs. The most commonly used mass spectrometry-based method for high-throughput histone PTM analysis is data-dependent acquisition (DDA). However, stochastic precursor selection and dependence on MS1 ions for quantification impede comprehensive interrogation of histone PTM states using DDA methods. To overcome these limitations, we utilized a data-independent acquisition (DIA) workflow that provides superior run-to-run consistency and postacquisition flexibility in comparison to DDA methods. In addition, we developed a novel DIA-based methodology to quantify isobaric, co-eluting histone peptides that lack unique MS2 transitions. Our method enabled deconvolution and quantification of histone PTMs that are otherwise refractory to quantitation, including the heavily acetylated tail of histone H4. Using this workflow, we investigated the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) on the global histone PTM state of human breast cancer MCF7 cells. A total of 62 unique histone PTMs were quantified, revealing novel SAHA-induced changes in acetylation and methylation of histones H3 and H4.