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首页> 外文期刊>Analytical chemistry >Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry
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Outer Membrane Protease OmpT-Based Strategy for Simplified Analysis of Histone Post-Translational Modifications by Mass Spectrometry

机译：基于外膜蛋白酶综合体的基于概述的策略，用于通过质谱分析组蛋白翻译后修饰的简化分析

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摘要

Histone modifications play an important role in regulating transcriptional gene expression and chromatin processes in eukaryotes. Increasing researches proved that aberrant post-translational modifications (PTMs) of histones is associated with many diseases. However, MS-based identification and quantification of histone PTMs are still challenging. Although classic chemical derivatization in conjunction with trypsin digestion is widely used for histone PTMs analysis in a bottom-up strategy, several side reactions have been observed in practice. In this work, outer membrane protease T (OmpT) was utilized as a protease for direct histone proteolysis and generated appropriate lengths of histone peptides for retention on reversed-phase chromatography. The powerful and unique tolerance of OmpT for modified lysines and arginines was demonstrated and can be quantitatively described for the first time, making it useful for detecting natural modifications. Using the optimized digestion conditions, we succeeded in identifying 121 histone marks from HEK293T cells, 42 of which were previously unreported. Additionally, histone H3 PTMs were quantitatively profiled in the KMS11 multiple myeloma cells and NSD2 selective knockout KMS11 cells, revealing that NSD2 was of high specificity on H3K36 dimethylation. Histone chemical derivatizations are not required in our strategy, showing a remarkable strength over the conventional trypsin-based workflow.
机译：组蛋白修饰在调节了真核生物中调节转录基因表达和染色质过程中的重要作用。越来越多的研究证明，组织的异常翻译后修饰（PTMS）与许多疾病有关。然而，基于MS的鉴定和组蛋白PTM的定量仍然具有挑战性。尽管与胰蛋白酶消化结合的经典化学衍生化广泛用于自下而上策略中的组蛋白PTMS分析，但在实践中已经观察到了几种副反应。在这项工作中，外膜蛋白酶T（OMPT）用作直接组蛋白蛋白水解的蛋白酶，并产生适当长度的组蛋白肽，用于保留反相色谱。对修饰的赖氨酸和精氨酸进行的EMPT的强大和独特的耐受性被证明，并且可以首次定量描述，使其可用于检测自然修改。使用优化的消化条件，我们成功地识别来自HEK293T细胞的121个组蛋白标记，其中42次以前未报告。另外，在KMS11多发性骨髓瘤细胞和NSD2选择性敲除KMS11细胞中定量地分析组蛋白H3 PTM，显示NS​​D2对H3K36二甲基化的特异性具有高特异性。我们的策略中不需要组蛋白化学衍生化，显示出对常规胰蛋白酶的工作流程的显着强度。

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  • 来源
    《Analytical chemistry》 |2020年第1期|共8页
  • 作者

    Hu Yajun; Zhang Lei; Chen Jiajia; Yao Jun; Jin Hong; Yang Pengyuan;

  • 作者单位

    Fudan Univ Shanghai Stomatol Hosp Shanghai 200433 Peoples R China;

    Fudan Univ Shanghai Stomatol Hosp Shanghai 200433 Peoples R China;

    Fudan Univ Shanghai Stomatol Hosp Shanghai 200433 Peoples R China;

    Fudan Univ Shanghai Stomatol Hosp Shanghai 200433 Peoples R China;

    Fudan Univ Shanghai Stomatol Hosp Shanghai 200433 Peoples R China;

    Fudan Univ Shanghai Stomatol Hosp Shanghai 200433 Peoples R China;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分析化学;
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