One-pot shotgun quantitative mass spectrometry characterization of histones.
One-pot shotgun quantitative mass spectrometry characterization of histones.
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DOI：
10.1021/pr900777e
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发表时间：
2009-11
影响因子：
4.4
通讯作者：
Garcia, Benjamin A.
中科院分区：
文献类型：
--
作者：
Plazas-Mayorca, Mariana D.;Zee, Barry M.;Young, Nicolas L.;Fingerman, Ian M.;LeRoy, Gary;Briggs, Scott D.;Garcia, Benjamin A.
关键词：
尽管越来越多的应用质谱（MS）来表征组蛋白的翻译后修饰（PTM），大多数现有的协议是不适合鲁棒地测量他们在一个高通量的定量方式。在这项工作中，我们扩大了目前的协议，并描述了改进的方法，定量自下而上的组蛋白和他们的PTM表征具有可比的灵敏度，但更高的吞吐量比标准的MS方法。这是通过首先绕过组蛋白的离线分级分离并直接使用来自典型核酸提取的总组蛋白来实现的。接下来，使用化学衍生化程序，结合稳定同位素标记在两个步骤的过程中，我们可以使用nanoLC-MS/MS定量比较样品。我们表明，我们的方法可以成功地检测17个组合H2 A/H2 B变体和超过25个组合组蛋白H3和H4 PTM在一个单一的MS实验。我们测试我们的方法，通过量化差异表达的组蛋白PTM从野生型酵母和甲基转移酶敲除菌株。这种改进的方法确定，在MS询问之前，不严格要求像通常进行的那样消耗时间和样品的单独组蛋白变体的离线HPLC或SDS-PAGE纯化。我们的协议显着简化组蛋白PTM的分析，并将允许在生物相关过程中以快速和定量的方式研究多个样品之间的差异表达的PTM。
Despite increasing applications of mass spectrometry (MS) to characterize post-translational modifications (PTMs) on histone proteins, most existing protocols are not properly suited to robustly measure them in a high-throughput quantitative manner. In this work, we expand on current protocols and describe improved methods for quantitative Bottom Up characterization of histones and their PTMs with comparable sensitivity, but much higher throughput than standard MS approaches. This is accomplished by first bypassing off-line fractionation of histone proteins and working directly with total histones from a typical nuclei acid extraction. Next, using a chemical derivatization procedure that is combined with stable-isotope labeling in a two-step process, we can quantitatively compare samples using nanoLC-MS/MS. We show that our method can successfully detect 17 combined H2A/H2B variants and over 25 combined histone H3 and H4 PTMs in a single MS experiment. We test our method by quantifying differentially expressed histone PTMs from wild-type yeast and a methyltransferase knockout strain. This improved methodology establishes that time and sample consuming off-line HPLC or SDS-PAGE purification of individual histone variants prior to MS interrogation as commonly performed is not strictly required. Our protocol significantly streamlines the analysis of histone PTMs and will allow for studies of differentially expressed PTMs between multiple samples during biologically relevant processes in a rapid and quantitative fashion.
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