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。我们展示了如何通过测量体内蛋白质对刺激的反应动力学来获得基因作用的功能图像，即一个人可以通过给予2H2O(一种安全的、非放射性的同位素)，然后测量其肽的2H标记来确定几乎任何蛋白质的合成速率(S)。由于2H2O是口服的，并且蛋白质组分析的灵敏度允许测量L的血浆体积，因此我们的方法提供了一种微创方法来测量饮食、药物和/或手术干预的反应。这项建议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开发。在展望广泛应用之前，必须先测试两个关键问题。我们的第一个目标是确定2H在体内水分和游离氨基酸之间的平衡。我们的第二个目标是通过确定肥胖的胰岛素抵抗表型和胰岛素抵抗的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。实验将集中在位于血浆蛋白质组两端的两种蛋白质，白蛋白和胰岛素，例如白蛋白具有相对较长的T1/2(在人类中为~10天)，并且存在于高浓度(~600微克分子/1000毫升)中，而胰岛素具有相对较短的T1/2(在人类中为~30分钟)，并且以低浓度(~100 pmol/1000毫升)存在。研究将对比极端生理动态平衡的啮齿动物模型，例如进食和禁食状态以及正常和胰岛素抵抗糖尿病。这些模型实验将引出挑战该方法在体内使用的主要因素。意义重大。首先，虽然我们使用传统的质谱计(与同位素比质谱仪相比)，但我们设计了新的数据集成例程，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“动态标记物”的潜力，可以预见许多研究领域的扩大。最后，由于2H2O可以在人体内使用，我们计划将这项技术应用于移植前和移植后的胰岛功能监测，本文将使用的技术开发和模型系统将促进这些未来的研究。 公共卫生相关声明：我们已经开发了一种新技术，该技术有助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.