FUNCTIONAL PROTEOMICS OF LEUKEMIC MARROW STROMAL CELLS

描述(由申请人提供)： 蛋白质组学需要全球基因表达分析，被认为是一种 无偏见的发现驱动(而不是假设驱动)方法 蛋白质表达分析。建议的长远目标 研究旨在确定人骨髓(BM)基质细胞在 正常和白血病的造血功能。这项建议旨在：(1)发扬 先前的重要发现表明，人类骨髓基质细胞代表 独特的多分化间充质祖细胞类型(MPC)， 共表达多种间充质系标记物。(2)最近申请 建立了从白血病BM基质细胞培养中纯化MPC的方法， 不含巨噬细胞和造血细胞。(3)调查MPC细胞特异性 蛋白质表达谱及其在造血系统中的变化 恶性肿瘤。(四)确立原则和实践的证明 与正常人和白血病人骨髓相关的高分辨率蛋白质组学 基质细胞。具体的实验设计包括 跟在后面。R21期：建立大幅面双向凝胶电泳系统 PAGE)，用于可重复分离MPC蛋白。制备双向PAGE蛋白 正常BM来源的MPC的MAP(未经处理和使用代表性药物处理 细胞因子)和来自典型白血病患者的MPC 条件(AML、CML、MM)。使用质谱学(MALDI-MS和/或纳米ESI MS/MS)，鉴定了大约200个差异表达的MPC蛋白(即 与2-D PAGE蛋白质图谱相比在强度上增加或减少 正常的、未受刺激的MPC)。在WWW上构建人类BM MPC-2DPAGE数据库 并在WORLD-2DPAGE下发布，并链接到当前存在的2-D 页面数据库。在平行研究中，确定了大约200个不同的 用一种独立的方法--同位素编码的亲和标签表达MPC蛋白 (ICAT)标记与LC/MS/MS相结合，基于理论等电点和分子量，构建了ICAT鉴定的MPC蛋白质的虚拟二维图谱，用于整合 转换成2-D页面数据库。R33阶段：将高吞吐量机器人添加到 R21期高分辨2-D-PAGE蛋白质组学的建立。在身份上进行标识 相对较大规模(约1800)的差异MPC蛋白 不同细胞因子刺激后在白血病中的表达 (AML、CML、MM)。通过以下方式促进对致病机制的理解 鉴定可能参与细胞信号转导的磷蛋白 小路。在平行研究中，通过ICAT鉴定约1800个MPC蛋白 方法。我们计划确定总共约2,000个功能相关的BM 每种方法提取基质细胞蛋白。更新WWW数据库，方法是将 表达发生病理变化的蛋白质。该数据库将是一个 为研究人类基础生物学的研究人员提供宝贵的资源 造血和白血病发生与临床血液学家/肿瘤学家 和病理学家一样。

DESCRIPTION (provided by applicant): Proteomics, which entails global gene expression analysis, is considered a nonbiased discovery-driven (as opposed to hypothesis-driven) approach to the analysis of protein expression. The long-term objective of the proposed research is to determine the role of human bone marrow (BM) stromal cells in normal and leukemic hematopoiesis. This proposal aims to: (1) Carry forward a critical previous discovery that shows that human BM stromal cells represent a unique pluridifferentiated mesenchymal progenitor cell type (MPC), coexpressing multiple mesenchymal lineage markers. (2) Apply a recently established method for purifying MPCs from leukemic BM stromal cell cultures, free of macrophages and hematopoietic cells. (3) Investigate MPC cell-specific protein expression profiles and how these profiles change in hematopoietic malignancies. (4) Establish the proof of principle and practice of high-resolution proteomics with relevance to normal and leukemic human BM stromal cells. Specific experimental design includes performance of the following. R21 Phase: Set up large-format 2-D gel electrophoretic system (2-D PAGE) for reproducible separation of MPC proteins. Prepare 2-D PAGE protein maps for normal BM-derived MPCs (untreated and treated with representative cytokines) and for MPCs derived from patients with representative leukemic conditions (AML, CML, MM). Using mass spectrometry (MALDI-MS and/or Nano ESI MS/MS), identify about 200 differentially-expressed MPC proteins (i.e., those that increased or decreased in intensity as compared to 2-D PAGE protein maps of normal, unstimulated MPCs). Construct a human BM MPC-2DPAGE database on WWW and publish it under WORLD-2DPAGE with links to the currently existing 2-D PAGE databases. In parallel studies, identify about 200 differentially expressed MPC proteins by an independent method, isotope-coded affinity tag (ICAT) labeling in conjunction with LC/MS/MS. Based on theoretical pI and MW, construct a "virtual" 2-D map of ICAT-identified MPC proteins for integration into 2-D PAGE database. R33 Phase: Add high-throughput robotics to the high-resolution 2-D PAGE proteomics established under R21 phase. Identify on a relatively large-scale (about 1,800) the MPC proteins that are differentially expressed following stimulation with different cytokines and in leukemias (AML, CML, MM). Facilitate understanding of the pathogenetic mechanisms by identifying the phosphoproteins potentially involved in cell signaling pathways. In parallel studies, identity about 1,800 MPC proteins by ICAT method. We plan to identify a total of about 2,000 functionally relevant BM stromal cell proteins by each method. Update the WWW database by including the proteins with pathologically altered expression. The database will be a valuable resource for researchers investigating the basic biology of hematopoiesis and leukemogenesis and for clinical hematologists/oncologists and pathologists alike.