Regulation of the Human Osteoblast Proteome

描述(由申请人提供)：此第一阶段SBIR应用程序检测成骨细胞蛋白表达的全球变化，以响应成骨生长因子。这项建议的长期目标是开发一种蛋白质组学方法来研究人类成骨细胞的功能，从而能够识别药物发现的治疗靶点。通过关注原代人类成骨细胞，这些靶点在生理上是相关的，最终对我们对骨质疏松等骨骼疾病的理解很重要。假设成骨生长因子刺激导致蛋白质表达/修饰的改变，这是蛋白质组学分析的结果。我们进一步假设，已知的骨活性生长因子的组合可以用作识别蛋白质靶点的概念验证方法。该项目的总体目标是发现潜在的细胞靶点，以刺激人类成骨细胞的发育；其长期目标是确定在骨疾病治疗中具有治疗价值的蛋白质靶点。这一阶段的SBIR重点展示了将蛋白质组学作为人类成骨细胞发育的发现工具的可行性，展示了细胞分离(成膜、胞浆和核蛋白)降低了蛋白质组分析的复杂性，并展示了通过结合二维凝胶电泳(2-DGE)和质谱学鉴定可以容易和快速地鉴定差异表达的蛋白质。这项拟议的工作代表着Velcura治疗公司和蛋白质研究服务公司之间的合作。Velcura治疗公司将执行提案中的生物和2DGE部分，蛋白质研究服务公司将进行质谱仪/蛋白质鉴定。这一建议代表了人类成骨细胞生物学、蛋白质组学分析和最先进的质谱学的创新结合，以确定由两种协同作用的成骨生长因子-转化生长因子-β和骨形成蛋白2差异调节的蛋白质。独特的是，细胞生物学使用在无血清介质中生长的原代人类成骨细胞作为三维组织样聚集体。因此，人类成骨细胞在更具生理性的环境中发育。据预测，Velcura/PRS的联合研究将产生几个不同的结果：1)建立用于发展人类成骨细胞的蛋白质图谱数据库的方案、格式和初始种群。2)提供了2-DGE平均图谱(单个人成骨细胞供体之间的)可以识别人成骨细胞的“激活状态”的概念证明。3)使用TGFbeta1和BMP-2产生概念证明，这些分子的蛋白质靶标可以通过2-DGE和MS/MS或LC/MS/MS识别4)识别75到525(取决于MS在每个蛋白点中分解的蛋白质的数量)蛋白质，这些蛋白质在膜、胞浆和核细胞部分中持续调节。

DESCRIPTION (provided by applicant): This Phase I SBIR application examines global changes in human osteoblast protein expression in responsive to osteogenic growth factors. The long-term goals of this proposal are to develop a proteomics approach to human osteoblast function that allows the identification of therapeutic targets for drug discovers. By focusing on primary human osteoblast, such targets are physiologically relevant, and ultimately important to our understanding of bone diseases such as osteoporosis. It is hypothesized that that osteogenic growth factor stimulation results in changes in protein expression/modification that are amenable to proteomic analysis. We further postulate that combinations of known bone-active growth factors can be used as a proof-of-concept approach to identify protein targets. The overall goal of this project is to discover potential cellular targets for stimulating human osteoblast development; its long-term objective is the identification of protein targets that are of therapeutic value in the treatment of bone diseases. This phase I SBIR focuses on demonstrating the feasibility of using proteomics as a discovery tool in human osteoblast development, on demonstrating that cellular fractionation (into membrane, cytosolic, and nuclear proteins) reduces the complexity of the proteomic analysis, and on showing that differentially expressed proteins can be readily, and rapidly identified by combined 2-dimensional gel electrophoresis (2-DGE) and mass spectrometry identification. The proposed work represents a collaboration between Velcura Therapeutics, Inc., who will perform the biological and 2DGE components of the proposal, and Proteomic Research Services, Inc. who will do the mass spectrometry/ protein identification. This proposal represents an innovative blend of human osteoblast cell biology, proteomic analyses, and state-of-the-art mass spectrometry to identify proteins differentially regulated by two synergistically interacting osteogenic growth factors, TGF-beta & BMP2. Uniquely, the cell biology uses primary human osteoblasts grown in serum-free media as 3-dimensional tissue-like aggregates. Thus, the human osteoblasts develop in a more physiological context. It is predicted that the joint Velcura/PRS investigations will result in several distinct outcomes: 1) Establish the protocol, format, and initial population of a protein map database for developing human osteoblasts. 2) Provide a proof-of-concept that 2-DGE Averaged Maps (among and between individual human osteoblasts cell donors) can identify "activation states" of human osteoblasts. 3) Produce a proof-of-concept, using TGFbeta1 and BMP-2, that the protein targets of these molecules can be identified by 2-DGE and MS/MS or LC/MS/MS. 4) Identify between 75 and 525 (depending on the number of proteins resolve by MS in each protein -spot) proteins that are consistently modulated in the membranes, cytosol, and nuclear cell fractions.