Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.
Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top-Down Proteomic Analysis.
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DOI：
10.1002/cpz1.26
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发表时间：
2021-03
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影响因子：
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通讯作者：
Young NL
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作者：
Holt MV;Wang T;Young NL
组蛋白是染色质的主要蛋白质成分，参与几乎所有的DNA模板化过程。组蛋白被各种染色质修饰机制大量地翻译后修饰。这些翻译后修饰(PTM)可被一系列“阅读器”蛋白识别，这些“阅读器”蛋白将额外的蛋白质招募到染色质上的特定位置，并对基因调控产生精确而强大的影响。每个PTM通常对转录产生积极或消极的影响，最近的研究表明，组蛋白PTMS在组合组蛋白编码中发挥作用：组蛋白PTMS结合在一起发挥作用，以实施精确的DNA模板调控。因此，有必要识别和理解蛋白质形式：明确定义的具有所有修饰组合的单一蛋白质分子。自上而下的蛋白质组学是目前识别和定量组蛋白蛋白形式的唯一可行的方法，质谱仪已经变得足够强大，能够以强健和高通量的方式进行这些定量分析。这些最近的创新使染色质研究的新的实验方向成为可能，但也引入了时间和其他限制。这导致我们开发了这里描述的协议，它增加了吞吐量，减少了样品需求，并保持了稳健的定量。虽然最初是为高通量定量自上而下的蛋白质组学而设计的，但这里描述的方案对于广泛的染色质生物学应用是有用的。从少量的细胞或组织开始，我们描述了两种非常快速和有效的核分离的基本方案，组蛋白的酸提取，以及组蛋白的高效液相分离成组蛋白家族。此外，我们还描述了组蛋白H4蛋白形式的定量自上而下的蛋白质组学分析。
Histones are the primary protein component of chromatin and are involved in virtually all DNA-templated processes. Histones are abundantly post-translationally modified by a variety of chromatin modifying machinery. These post-translational modifications (PTMs) are recognized by a range of ‘reader’ proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: histone PTMs function in combination to exert precise DNA-templated regulation. Thus, there is a need to identify and understand proteoforms: unambiguously defined single protein molecules with all combinations of modifications. Top-down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high-throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocol described here, which increases throughput, reduces sample requirements, and maintains robust quantitation. Although originally designed for high-throughput quantitative top-down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two Basic Protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high-performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top-down proteomic analysis of histone H4 proteoforms.