Multiplexing Protein Analysis Core

多重蛋白质分析核心

• 批准号: 10424599
• 负责人: Michael T Kinter
• 金额: $ 18.68万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10424599
• 关键词: Acetylation; Affinity; Aging; Animal Model; Animals; Biochemical Pathway; Biogenesis; Biological Assay; Biology; Biology of Aging; Caenorhabditis elegans; Cell Proliferation; Cells; Citric Acid Cycle; Communities; Complex; Consult; Core Facility; DNA; DNA biosynthesis; Data Analyses; Deuterium; Deuterium Oxide; Development; Disease; Dissociation; Drosophila genus; Freezing; Funding; Genome; Geroscience; Gluconeogenesis; Glycolysis; Goals; Half-Life; Immobilization; Individual; Ions; Kinetics; Label; Laboratories; Mass Fragmentography; Mass Spectrum Analysis; Measurement; Measures; Metals; Methods; Modeling; Modification; Molecular; Monitor; Mus; Nucleotides; Oklahoma; Peptides; Phosphorylation; Post-Translational Protein Processing; Preparation; Procedures; Process; Protein Analysis; Protein Biosynthesis; Proteins; Proteome; Proteomics; RNA; RNA chemical synthesis; Rattus; Reaction; Regulation; Research; Research Design; Research Personnel; Resolution; Resources; Ribosomes; Running; Sampling; Services; Shock; Stable Isotope Labeling; Sum; System; Tissue Model; Tissues; Tracer; Trypsin; Western Blotting; Yeasts; age related; base; cell type; design; experience; experimental study; flexibility; insight; instrument; interest; protein degradation; proteostasis; sample collection; selected ion monitoring; stable isotope; tandem mass spectrometry; tool; ultra high resolution

The goal of the Multiplexing Protein Analysis Core (MPAC) is to provide users with specialized tools to determine the dynamic regulation of the proteome. As in the previous cycle, our primary service includes the rigorous, sensitive and precise quantification of panels of proteins relevant to the basic biology of aging and age-related disease. For this cycle, we expand the capabilities of the core by adding stable isotope tracer experiments with deuterium oxide (D2O) to measure the turnover of proteins. Although core facilities that offer discovery-based proteomics are relatively common, only a few cores offer these targeted methods. Further, the Core offers these assays in panels that interrogate specific biochemical pathways important in aging and can design new assays and panels on request for any protein from any animal with a sequenced genome. In addition, the Core can use its targeted approaches for post-translational modifications such as phosphorylation. There are also relatively few laboratories with the expertise to measure protein turnover rates using stable isotopes. Measuring synthetic rates with tracers requires proper study design, mass spectrometry with appropriate sample preparation and analysis, and correct interpretation of data. The advantages of D2O for Core users are significant. Specifically, it is cheap, highly sensitive, flexible, biologically inert, lends itself to long-term labeling, and can be used to measure the synthesis of a variety of molecules. The combination of D2O labeling and targeted proteomics in one sample allows users to understand changes in the content of individual proteins, the turnover processes that drive the changes, and mechanisms such as cell proliferation and ribosomal biogenesis that contribute to these changes. Finally, the analyses provided by the core are made on frozen samples, facilitating ease of sample collection for outside users. The Core proposes two specific aims: 1) Develop and apply high throughput multiplexed protein quantification for panels of proteins, including post-translational modifications, in experimental systems used by Geroscience investigators, including mice, rats, fruit flies, C.elegans, and yeast, and 2) Use stable isotope labeling and analysis in combination with multiplexed protein quantification to measure turnover of individual proteins as well as processes that contribute to the regulation of protein abundance. To accomplish the aims, the MPA Core uses selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) in tandem mass spectrometry systems or high-resolution accurate mass (HRAM) selected ion monitoring (SIM) in an orbitrap mass spectrometry system, as well as GC-MS based analysis of supportive measurements. The ability to adapt these procedures to multiple cell types, tissues, and model organisms make the MPA Core a significant resource for the aging research community.

多重蛋白质分析核心(MPAC)的目标是为用户提供专门的工具来 确定蛋白质组的动态调节。与前一个周期一样，我们的主要服务包括 严格、灵敏和精确地量化与衰老和基础生物学有关的蛋白质组 与年龄相关的疾病。对于这个周期，我们通过添加稳定的同位素示踪剂来扩展核心的能力 用氧化氢(D2O)测量蛋白质周转率的实验。尽管核心设施提供 基于发现的蛋白质组学是相对常见的，只有少数核心提供这些有针对性的方法。此外， CORE在小组中提供了这些分析，这些小组询问在衰老中重要的特定生化途径，并可以 根据要求设计新的分析方法和面板，以从任何具有已测序基因组的动物中提取任何蛋白质。在……里面 此外，核心可以使用其有针对性的方法进行翻译后修改，如 磷酸化。拥有测量蛋白质周转率的专业技术的实验室也相对较少。 使用稳定的同位素。用示踪剂测量合成速率需要适当的研究设计，质谱学 适当的样品准备和分析，以及对数据的正确解释。D2O的优势 对于核心用户来说意义重大。具体地说，它廉价、高度敏感、灵活、生物惰性，适合于 长期标记，并可用于测量各种分子的合成。这两种技术的结合 在一个样本中进行D2O标记和靶向蛋白质组学使用户能够了解 单个蛋白质，驱动变化的周转过程，以及细胞增殖等机制 以及导致这些变化的核糖体生物发生。最后，核心提供的分析是 在冷冻样品上制作，便于外部用户轻松采集样品。核心提出了两项建议 具体目标：1)开发和应用用于蛋白质组的高通量多路复用蛋白质定量， 包括翻译后修饰，在老年科学研究人员使用的实验系统中， 包括小鼠、大鼠、果蝇、线虫和酵母，以及2)使用稳定同位素标记和分析 与多重蛋白质定量相结合来测量单个蛋白质的周转以及 有助于调节蛋白质丰度的过程。为了实现这些目标，MPA核心使用了 串联质谱仪中的选择反应监测(SRM)和平行反应监测(PRM) 系统或高分辨率精确质量(HRAM)选择离子监测(SIM)在轨道碰撞质量中 光谱系统，以及基于GC-MS的支持性测量分析。适应能力 这些针对多种细胞类型、组织和模式生物的程序使MPA核心成为一个重要的 老龄化研究社区的资源。