Quantifying Histone Modifications Associated with Age using Mass Spectrometry

使用质谱定量与年龄相关的组蛋白修饰

• 批准号: 8521027
• 负责人: Elisabeth M Hersman
• 金额: $ 3.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521027
• 关键词: Acetylation; Age; Aging; Biological; Biological Markers; Biology; Biology of Aging; Blood; Caloric Restriction; Cell Count; Cell Culture Techniques; Cell Density; Cell Survival; Cell physiology; Cells; Chemicals; Chromatography; Cues; DNA; Data; Data Set; Dissociation; Drug Targeting; Electron Transport; Embryo; Enzymes; Epigenetic Process; Equilibrium; Eukaryota; Genetic Materials; Glucose; Harvest; Health; Histones; Human; Knock-out; Label; Lead; Libraries; Life; Longevity; Mass Spectrum Analysis; Measures; Methods; Methylation; Modification; Molecular Biology; Monitor; Morphologic artifacts; Mus; Names; Optics; Organism; Pathway interactions; Pattern; Peptides; Peripheral Blood Mononuclear Cell; Phenotype; Phosphorylation; Play; Post-Translational Protein Processing; Productivity; Proteins; Proteomics; Relative (related person); Research; Research Personnel; Resolution; Role; Sampling; Signaling Protein; Site; Techniques; Validation; Western Blotting; Yeasts; Youth; age effect; base; cell type; density; high risk; histone modification; improved; interest; kidney cell; mass spectrometer; multiple reaction monitoring; novel; protein purification; repaired; research study; stem; trend; validation studies

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.

描述（由申请人提供）：衰老改变了体内所有细胞通路网络的平衡。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非常重要，但可以说同样重要的是表观遗传蛋白质，它控制DNA的哪些部分可用并转录。这些表观遗传蛋白中的一组被称为组蛋白，其可以通过乙酰化、甲基化和磷酸化来修饰。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。为了分析组蛋白上的修饰，使用传统的非定量但灵敏和精确的方法，质谱法。氘代乙酰化是一种同位素标记策略，可以更灵敏地识别和定量位点特异性修饰。实验分为三个部分。1)开发一种互补的质谱方法（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。预计这将使感兴趣的残基的覆盖率从约60%增加到95%。将样品氘代乙酰化至完全，切成可分析的片段，然后用轨道阱高分辨率质谱仪进行分析。2)分析在低葡萄糖（卡路里限制）或高葡萄糖（非限制）培养基中生长的六个年龄的酵母中存在的每种修饰的种类和数量。热量限制（CR）已被证明可以增加酵母，小鼠和现在人类的平均寿命和最大年龄。实验将使用转基因酵母，其中含有HIS标记的组蛋白，这些组蛋白很容易使用Ni-NTA色谱法纯化。三次生物学重复将与 酵母活力研究（以监测有效年龄）。初步结果显示，随着年龄的增长，变化很小，在热量限制期间，变化很大。一些指示老化效应的残留物列在通过蛋白质印迹法进行的老化研究中的残留物中，证实了初步结果。3)使用三种策略验证CR和老化条件下生物学感兴趣的一种特定修饰的丰度：另一种定量质谱法（多反应监测）、酵母酶敲除菌株和蛋白质印迹以确定组蛋白修饰是否减少以及对老化谱的影响，两种人细胞类型（HEK 293稳定细胞培养物和来自人血的新鲜外周血单核细胞）将用于使用原始质谱法确认修饰。内源性组蛋白将通过酸性纯化方法进行纯化。这项令人兴奋的新研究将全面了解组蛋白如何与衰老和热量限制有关，为提高组蛋白研究的生产力铺平了上级工作流程，并为衰老生物学研究人员提供了新的研究目标。