Quantifying Histone Modifications Associated with Age using Mass Spectrometry

描述(申请人提供)：衰老改变了体内所有细胞的通路网络的平衡。遗传物质的活动是最持久和最广泛的细胞功能之一。虽然DNA显然很重要，但一些同样重要的参与者是表观遗传蛋白，它们控制着DNA的哪些部分可用并进行转录。其中一组表观遗传蛋白称为组蛋白，它可以通过乙酰化、甲基化和磷酸化来修饰，仅举几例。蛋白质上的这些化学修饰标志着DNA组织、复制和修复的变化。为了描述组蛋白上的修饰，使用了一种传统的非定量但灵敏和精确的方法-质谱学。去氢乙酰化是一种同位素标记策略，它可以更灵敏地鉴定和量化特定部位的修饰。拟议的实验包括三个部分。1)建立一种互补的质谱学方法(电子转移解离)，以增加所有四种组蛋白的组蛋白序列覆盖率。预计这将把感兴趣的残留物的覆盖率从大约60%提高到95%。样品经过去氢乙酰化，切成可分析的碎片，然后用Orbitrap高分辨率质谱仪进行分析。2)描述在低糖(卡路里限制)或高糖(非限制)培养基中生长的六个年龄段的酵母，每种修饰存在的程度和程度。卡路里限制(CR)已被证明可以延长酵母、小鼠和现在的人类的平均寿命和最长寿命。实验将使用转基因酵母，这种酵母含有His标记的组蛋白，使用Ni-NTA层析很容易纯化。三个生物复制体将与 酵母活性研究(以监测有效年龄)。初步结果显示，随着年龄的增长变化不大，而在热量限制的过程中变化很大。通过蛋白质印迹分析，在老化研究的残基中列出了一些指示老化效应的残基，证实了初步结果。3)为了验证在CR和老化条件下生物感兴趣的一种特定修饰的丰度，使用三种策略：替代定量质谱法(多反应监测)、酵母酶敲除菌株和Western blotting以确定组蛋白修饰是否减少以及对衰老模式的影响，并将使用两种人类细胞类型(HEK293稳定细胞培养和新鲜人血单个核细胞)来确认使用原始质谱法进行的修饰。内源性组蛋白将通过酸性提纯方法进行提纯。这项令人兴奋的新研究将提供组蛋白如何与衰老和卡路里限制有关的全貌，为提高组蛋白研究的生产率铺平了一条优越的工作流程，并为老年生物学研究人员提供了新的研究目标。 与公共健康相关：组蛋白是在所有生物中组织DNA的蛋白质，被认为通过化学修饰参与了衰老生物学的机制。更深入地了解组蛋白在衰老中的作用可能会导致药物靶点将健康延长到老年。我们建议使用一种新的质谱学技术来分析酵母老化、人类细胞培养和新鲜人类外周血单核细胞中组蛋白的变化。

DESCRIPTION (provided by applicant): Aging changes the balance of pathway networks of all the cells in the body. The activity of the genetic material is one of the most permanent and widespread cellular function. While DNA is very obviously important, some arguably equally important players are the epigenetic proteins which control what parts of the DNA are available and transcribing. One group of these epigenetic proteins is called histones, which can be modified by acetylation, methylation, and phosphorylation to name a few. These chemical modifications on the proteins signal changes in DNA organization, replication, and repair. To profile the modifications on histones, a traditionally non-quantitative but sensitive and precise method, mass spectrometry, is used. Deuteroacetylation, an isotopic labeling strategy, allows even more sensitive identification and also quantification of site-specific modifications. The proposed experiments have three parts. 1) To develop a complementary mass spectrometric method (electron transfer dissociation) to increase histone sequence coverage of all four histones. It is expected that this will increase coverage of interesting residues from approximately 60% to 95%. The sample is deuteroacetylated to completion, cut into analyzable fragments, then analyzed with an orbitrap high resolution mass spectrometer. 2) To profile which and how much of each modification is present for six ages of yeast grown in low glucose (calorie restricting) or high glucose (non-restricting) media. Calorie restriction (CR) has been shown to increase the average lifespan and maximum age of yeast, mice and now humans. Experiments will use genetically modified yeast which contain HIS-tagged histones that are easily purified using Ni-NTA chromatography. Triplicate biological replicates will be correlated to a yeast viability study (to monitor effective age). Preliminary results show slight with age, and drastic changes during calorie-restriction. Some residues indicating aging effects list amongst the residues featured in aging studies by western blotting, confirming the preliminary results. 3) To validate the abundance of one particular modification of biological interest in the CR and aging conditions using three strategies: an alternative quantitative mass spectrometric method (multiple reaction monitoring), yeast enzyme knock-out strains and western blotting to determine if the histone modification decreases and what the effect is on the aging profile, and two human cell types (HEK293 stable cell culture and fresh peripheral blood mononuclear cell from human blood) will be used to confirm the modification using the original mass spectrometric method. Endogenous histones will be purified by an acidic purification method. This exciting new research will provide a full picture of how histones are implicated in aging and calorie restrictio, paving a superior workflow to improve productivity of histone research and provide new targets for aging biology researchers to study. PUBLIC HEALTH RELEVANCE: Histones, the proteins which organize DNA in all living things, are thought to be involved in the mechanism of aging biology through chemical modification. A deeper understanding of the histone role in aging could lead to drug targets to extend health into old age. We propose to use a new mass spectrometric technique to analyze histone changes throughout yeast aging, in human cell culture, and in fresh human peripheral blood mononuclear cell.