Multiplexing Protein Analysis Core

多重蛋白质分析核心

基本信息

批准号：
10044527
负责人：
Michael T Kinter
金额：
$ 17.68万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
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/10044527
关键词：
Acetylation Affinity Aging Animal Model Animals Biochemical Pathway Biogenesis Biological 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

项目摘要

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.

多路复用蛋白分析核心（MPAC）的目标是为用户提供专业工具确定蛋白质组的动态调节。与上一个周期一样，我们的主要服务包括严格，敏感和精确的蛋白质面板与衰老和衰老的基本生物学相关的蛋白质面板与年龄有关的疾病。对于此周期，我们通过添加稳定的同位素示踪剂来扩展核心的功能用氧化氘（D2O）进行实验，以测量蛋白质的周转率。虽然提供的核心设施基于发现的蛋白质组学相对常见，只有少数核心提供这些目标方法。此外，核心在面板中提供这些测定法，以审问特定的生化途径在衰老中很重要，并且可以根据要求使用任何具有测序基因组的动物的蛋白质设计新的测定法和面板。在此外，核心可以使用其目标方法进行翻译后修改，例如磷酸化。具有专业知识的实验室相对较少的实验室来衡量蛋白质更新率使用稳定的同位素。用示踪剂测量合成率需要适当的研究设计，质谱法通过适当的样本准备和分析，并正确解释数据。 D2O的优势对于核心用户很重要。具体而言，它便宜，高度敏感，灵活，生物学上的惰性，使自己适合长期标记，可用于测量各种分子的合成。结合 D2O标记和一个样本中的靶向蛋白质组学允许用户了解内容的变化单个蛋白质，驱动变化的周转过程以及诸如细胞增殖之类的机制和核糖体的生物发生有助于这些变化。最后，核心提供的分析是用冷冻样品制作，促进外部用户的易于收集样品。核心提出了两个具体目的：1）开发和应用高通量多路复用的蛋白质定量，用于蛋白质面板，包括翻译后修改，在Geroscience研究者使用的实验系统中，包括小鼠，大鼠，水果蝇，C.果皮和酵母，以及2）在中使用稳定的同位素标记和分析与多重蛋白质定量结合，以测量单个蛋白质的周转率有助于调节蛋白质丰度的过程。为了实现目标，MPA核心使用串联质谱法中选定的反应监测（SRM）和平行反应监测（PRM）系统或高分辨率精确质量（HRAM）在Orbitrap质量中选定的离子监测（SIM）光谱系统以及基于GC-MS的支持测量分析。适应能力这些对多种细胞类型，组织和模型生物的程序使MPA核心成为显着的老龄化研究界的资源。