Enabling studies of proteome dynamics

促进蛋白质组动力学研究

• 批准号: 7788183
• 负责人: Takhar Kasumov
• 金额: $ 19.43万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788183
• 关键词: Acute; Affect; Albumins; Amino Acids; Area; Biological Assay; Biological Models; Biopsy; Blood Plasma Volume; Body Water; Development; Diabetes Mellitus; Diagnosis; Disease; Ensure; Fasting; Functional Imaging; Future; Gene Expression; Genes; Glucose tolerance test; Health Status; Homeostasis; Human; Ingestion; Insulin; Insulin Resistance; Investigation; Isotope Labeling; Isotopes; Kinetics; Label; Mass Fragmentography; Mass Spectrum Analysis; Measurement; Measures; Methods; Modeling; Molecular Profiling; Monitor; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Operative Surgical Procedures; Patient Monitoring; Peptides; Phenotype; Physiological; Plasma; Protein Biosynthesis; Protein Dynamics; Proteins; Proteome; Proteomics; Radioisotopes; Rattus; Reproducibility; Research; Research Infrastructure; Rodent Model; Route; Stimulus; Technology; Testing; Tissues; Transplantation; Work; base; data integration; diabetic; feeding; improved; in vivo; innovative technologies; mass spectrometer; minimally invasive; new technology; novel; pancreatic islet function; protein metabolism; public health relevance; research study; response; software development; technology development

DESCRIPTION (provided by applicant): Proteomic investigations are limited, in that, they typically yield information regarding static gene expression profiles. We demonstrate how to obtain a functional image of gene action by measuring protein dynamics in response to stimuli in vivo, i.e. one can determine the synthesis rate of virtually any protein by administering 2H2O (a safe, non-radioactive isotope) and then measuring the 2H-labeling of its peptide(s). Since 2H2O is given orally and the sensitivity of the proteomic assays allows measurements on ¿l volumes of plasma, our method offers a minimally-invasive means of measuring the response to a dietary, pharmacological and/or surgical intervention. The objective of this proposal is to enable the routine application of our innovative technology for studying the development of proteome expression profiles. Two pivotal issues must be tested before one can envision wide-spread application. Our first aim is to determine the equilibration of 2H between body water and free amino acids. Our second aim is to test the applicability of 2H2O -proteomics by determining the influence of an obese insulin resistant phenotype and insulin resistant diabetes on protein synthesis in vivo. Experiments will focus on two proteins that lie at extreme ends of the plasma proteome, albumin and insulin, e.g. albumin has a relatively long t1/2 (~ 10 days in a human) and is present in high concentration (~ 600 umol per 1000 ml) whereas insulin has relatively short t1/2 (~ 30 min in a human) and is present in low concentration (~ 100 pmol per 1000 ml). Studies will contrast rodent models of extreme physiological homeostasis, e.g. fed vs. fasted states and normal vs. insulin resistant ¿ diabetes. These model experiments will draw out the main factors that challenge the use of the method in vivo. Significance. First, although we use conventional mass spectrometers (as compared to isotope ratio mass spectrometers), we have devised novel data integration routines to that yield "high precision" measurements of isotope labeling. Second, given the existing proteomics infrastructure in the world, and the potential of using "dynamic markers" of health/disease, one can envision the expansion of numerous areas of research. Last, since 2H2O can be administered in humans we plan to apply this technology for monitoring pancreatic islet function pre- and post-transplant, the technology development and model systems to be used herein will facilitate those future investigations. Public Health Relevance Statement: We have developed a new technology that is useful in diagnosing and monitoring patient health status, this relies on measurements of protein metabolism. We have outlined the essential studies that are required before one can envision wide-spread application.

描述（由申请人提供）：蛋白质组学研究是有限的，因为它们通常产生有关静态基因表达谱的信息。我们演示了如何通过测量体内刺激响应的蛋白质动力学来获得基因作用的功能图像，即通过施用 2H2O（一种安全的非放射性同位素），然后测量其肽的 2H 标记，可以确定几乎任何蛋白质的合成率。由于 2H2O 是口服给药，并且蛋白质组学测定的灵敏度允许测量 1 体积的血浆，因此我们的方法提供了一种微创方法来测量对饮食、药理学和/或手术干预的反应。该提案的目的是使我们的创新技术能够常规应用来研究蛋白质组表达谱的开发。在设想广泛应用之前，必须测试两个关键问题。我们的首要目标是确定体内水和游离氨基酸之间的 2H 平衡。我们的第二个目标是通过确定肥胖胰岛素抵抗表型和胰岛素抵抗糖尿病对体内蛋白质合成的影响来测试 2H2O -蛋白质组学的适用性。实验将集中于位于血浆蛋白质组最末端的两种蛋白质：白蛋白和胰岛素。白蛋白的 t1/2 相对较长（人体约 10 天），且浓度较高（每 1000 毫升约 600 umol），而胰岛素的 t1/2 相对较短（人体约 30 分钟），且浓度较低（每 1000 毫升约 100 pmol）。研究将对比极端生理稳态的啮齿动物模型，例如进食状态与禁食状态以及正常状态与胰岛素抵抗 — 糖尿病。这些模型实验将找出挑战该方法在体内使用的主要因素。意义。首先，虽然我们使用传统的质谱仪（与同位素比质谱仪相比），但我们设计了新颖的数据集成例程，以产生同位素标记的“高精度”测量。其次，考虑到世界上现有的蛋白质组学基础设施，以及使用健康/疾病“动态标记”的潜力，人们可以预见许多研究领域的扩展。最后，由于 2H2O 可以用于人类，我们计划应用该技术来监测移植前和移植后的胰岛功能，本文中使用的技术开发和模型系统将有助于未来的研究。 公共卫生相关性声明：我们开发了一种可用于诊断和监测患者健康状况的新技术，该技术依赖于蛋白质代谢的测量。我们概述了在设想广泛应用之前需要进行的基本研究。