Regulation of the Human Osteoblast Proteome

人类成骨细胞蛋白质组的调控

• 批准号: 6735772
• 负责人: Kathryn J MacLeod
• 金额: $ 22.37万
• 依托单位: VELCURA THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6735772
• 关键词: bone development; cell proliferation; clinical research; growth factor; human tissue; mass spectrometry; matrix assisted laser desorption ionization; method development; molecular biology information system; osteoblasts; protein quantitation /detection; protein structure function; proteomics; transforming growth factors; two dimensional gel electrophoresis

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.

描述（由申请人提供）：这项I期SBIR申请研究了人类成骨细胞蛋白表达对成骨生长因子的响应的全球变化。这项建议的长期目标是发展一种蛋白质组学方法来研究人类成骨细胞的功能，从而确定药物发现的治疗靶点。通过关注原代人成骨细胞，这些靶点在生理上是相关的，并且最终对我们理解骨质疏松症等骨病很重要。据推测，成骨生长因子刺激导致蛋白质表达/修饰的变化，这可用于蛋白质组学分析。我们进一步假设，已知骨活性生长因子的组合可以作为一种概念验证方法来识别蛋白质靶点。该项目的总体目标是发现刺激人类成骨细胞发育的潜在细胞靶点；其长期目标是鉴定在骨病治疗中具有治疗价值的蛋白靶点。第一阶段SBIR的重点是证明在人类成骨细胞发育中使用蛋白质组学作为发现工具的可行性，证明细胞分离（分为膜蛋白、细胞质蛋白和核蛋白）降低了蛋白质组学分析的复杂性，并表明通过结合二维凝胶电泳（2-DGE）和质谱鉴定可以方便、快速地鉴定差异表达蛋白。提议的工作是Velcura Therapeutics， Inc.和Proteomic Research Services， Inc.之间的合作，前者将执行提议的生物和2DGE组件，后者将进行质谱/蛋白质鉴定。该提案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分析的创新融合，以鉴定由两种协同作用的成骨生长因子tgf - β和BMP2差异调节的蛋白质。独特的是，细胞生物学使用在无血清培养基中生长的原代人成骨细胞作为三维组织样聚集体。因此，人类成骨细胞是在生理环境下发育的。预计Velcura/PRS联合研究将产生以下几个不同的结果：1)建立用于发育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)提供一个概念证明，2- dge平均图谱（在个体人类成骨细胞供体之间和之间）可以识别人类成骨细胞的“激活状态”。3)利用TGFbeta1和BMP-2进行概念验证，证明这些分子的蛋白靶点可以通过2-DGE和MS/MS或LC/MS/MS进行鉴定。4)鉴定在膜、细胞质和核细胞组分中一致调节的75到525个（取决于每个蛋白点中质谱解析的蛋白质数量）蛋白质。