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Characterization of histones and their posttranslational modifications by mass spectrometry.

机译：通过质谱表征组蛋白及其翻译后修饰。

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摘要

In this dissertation, mass spectrometry-based approaches were developed to characterize histones and their posttranslational modifications (PTMs). Histones are the primary protein component of chromatin. Histone tails are subject to a variety of PTMs including acetylation, methylation, and phosphorylation. These PTMs play critical roles in the regulation of chromatin remodeling and gene transcription. Therefore there is a need for better approaches to characterize histones and their PTMs. Yeast are chosen as the model for my studies due to many of their beneficial properties.;Methods for yeast histone preparation, separation, and LC-MS were developed and described in Chapter 2. Through extensive washing of nuclei prior to lysis and acid extraction, yeast histones were extracted from yeast whole cells with a high yield and purity. The isolated yeast histones were then separated by use of 16.5% Tris-tricine-SDS gels and characterized by LC-MS with trifluoroacetic acid (TFA) as the ion-pairing agent with minimal TFA effects. By using the methods developed in Chapter 2, histone acetylation and methylation patterns in yeast were studied (Chapter 3). We found that acetylation and trimethylation have different effects on retention time in reversed-phase liquid chromatography. The former shifts retention time, while the latter does not. By using retention time, acetylation and trimethylation were unambiguously distinguished even on a low-resolution mass spectrometer. In Chapter 4, we examined five JmjC domain-containing proteins (Ecm5, Gis1, Rph1, Jhd1, and Jhd2) in yeast for demethylase activities. Lysine methylation used to be considered irreversible. By using mass spectrometry-based approaches, we found that these proteins (except for Ecm5) displayed demethylase activity. In particular, Rph1 exhibited specificity toward H3K36 di- and trimethylation and Jhd2 had specificity toward H3K4 trimethylation. Cross-talk between methylation and acetylation was assessed in Chapter 5 by alterating the expression of the JmjC proteins. Our results show that H3 acetylation changes significantly in all strains expect for ecm5Delta. LC-MS/MS results indicate that Gis1 and Jhd1 facilitate K56Ac, while Rph1 decreases the level of K56Ac. Taking into consideration that the JmjC proteins are histone demethylases, this study implies that H3 methylation is correlated with acetylation on H2B1, H2B2, and H3.
机译：本文研究了基于质谱的方法来表征组蛋白及其翻译后修饰（PTM）。组蛋白是染色质的主要蛋白质成分。组蛋白尾巴要经历各种PTM，包括乙酰化，甲基化和磷酸化。这些PTM在染色质重塑和基因转录的调控中起着关键作用。因此，需要一种更好的方法来表征组蛋白及其PTM。酵母因其许多有益特性而被选为我的研究模型。第二章开发并描述了酵母组蛋白的制备，分离和LC-MS方法。通过在裂解和酸提取之前对核进行大量洗涤，从酵母全细胞中以高产率和高纯度提取酵母组蛋白。然后，通过使用16.5％的Tris-tricine-SDS凝胶分离分离的酵母组蛋白，并以三氟乙酸（TFA）作为离子配对剂的LC-MS进行表征，同时具有最小的TFA效应。通过使用第2章中开发的方法，研究了酵母中组蛋白的乙酰化和甲基化模式（第3章）。我们发现乙酰化和三甲基化在反相液相色谱中对保留时间有不同的影响。前者改变保留时间，而后者则不改变。通过使用保留时间，即使在低分辨率质谱仪上也能清楚地区分乙酰化和三甲基化。在第4章中，我们检查了酵母中5种含JmjC域的蛋白（Ecm5，Gis1，Rph1，Jhd1和Jhd2）的脱甲基酶活性。赖氨酸甲基化过去被认为是不可逆的。通过使用基于质谱的方法，我们发现这些蛋白质（Ecm5除外）显示出脱甲基酶活性。特别是，Rph1对H3K36二甲基和三甲基化表现出特异性，而Jhd2对H3K4三甲基化具有特异性。第5章通过改变JmjC蛋白的表达来评估甲基化和乙酰化之间的串扰。我们的结果表明，在所有预期为ecm5Delta的菌株中，H3乙酰化的变化都很大。 LC-MS / MS结果表明，Gis1和Jhd1促进K56Ac，而Rph1降低K56Ac的水平。考虑到JmjC蛋白是组蛋白脱甲基酶，这项研究表明H3甲基化与H2B1，H2B2和H3上的乙酰化相关。

著录项

  • 作者

    Yang, Lanhao.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Chemistry Inorganic.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 264 p.
  • 总页数 264
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 无机化学;
  • 关键词

  • 入库时间 2022-08-17 11:38:30

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