Enabling studies of proteome dynamics

描述（由申请人提供）：蛋白质组学研究是有限的，因为它们通常产生关于静态基因表达谱的信息。我们演示了如何通过测量蛋白质对体内刺激的响应动力学来获得基因作用的功能图像，即通过施用2 H2O（一种安全的非放射性同位素），然后测量其肽的2 H标记，可以确定几乎任何蛋白质的合成速率。由于2 H2O是口服给药的，并且蛋白质组学测定的灵敏度允许对1/4体积的血浆进行测量，因此我们的方法提供了一种测量对饮食、药理学和/或手术干预的反应的微创方法。该提案的目的是使我们的创新技术能够常规应用于研究蛋白质组表达谱的发展。在设想广泛应用之前，必须对两个关键问题进行测试。我们的第一个目标是确定体内水分和游离氨基酸之间的2 H平衡。我们的第二个目的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2 H2O-蛋白质组学的适用性。实验将集中在两种位于血浆蛋白质组末端的蛋白质，白蛋白和胰岛素，例如，白蛋白具有相对较长的t1/2（在人体中约10天），并以高浓度存在（约600 μ mol/1000 ml），而胰岛素的t1/2相对较短（人体约30 min），且浓度较低（约100 pmol/1000 ml）。研究将对比极端生理稳态的啮齿动物模型，例如进食与禁食状态以及正常与胰岛素抵抗性糖尿病。这些模型实验将引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），我们已经设计了新的数据集成程序，以产生同位素标记的“高精度”测量。其次，鉴于世界上现有的蛋白质组学基础设施，以及使用健康/疾病的“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2 H2O可用于人体，我们计划将该技术应用于监测移植前后的胰岛功能，本文所用的技术开发和模型系统将促进这些未来的研究。 公共卫生相关性声明：我们开发了一种新技术，可用于诊断和监测患者的健康状况，这依赖于蛋白质代谢的测量。我们已经概述了在人们可以设想广泛应用之前所需的基本研究。

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.