CORE--PROTEOMICS

核心E的主要目标是识别新的生物标记物，旨在表征肺 重度肺动脉高压(PAH)患者的血管和右室重构 ART蛋白质组学技术。具有核心B-D的核心E支持该SCCOR的项目1、3、4和5，包括 基于四个战略的基本实验服务。首先，核心，使用广泛的蛋白质组学 分析，将表征肺和右室肌细胞蛋白质组的变化 开发实验和人类多环芳烃(项目1、3、4和5以及岩心C和D)。这 该战略将得到JHU内现有的蛋白质组基础设施和既定专业知识的支持。 NHLBI蛋白质组学中心和JHU Bayview蛋白质组学中心。深入分析，使用多个 蛋白质组平台，包括二维差示成像电泳法、二维液相法 层析、O16/O18同位素标记、相对和绝对等压标记及定量 (ITRAQTM)、MALDI-TOF和液相色谱/质谱仪将被用于最大化蛋白质组 覆盖范围，同时允许对蛋白质异构体和翻译后修饰进行表征。第二, 核心将提供一个集中设施，以分析、汇编和关联候选人的量化水平 生物标记物，包括发现平台产生的新标记物以及其他潜在的 硬皮病相关性PAH相关候选生物标志物(如血管内皮生长因子、HIMF、基质金属蛋白酶-9和颗粒酶B) Beckman A2多重免疫分析将用于评估血清样本，而化学喷墨 打印机(芯片，蛋白质组系统)将用于我们的目标方法，以确认表达差异 来自使用肺和右室组织的基因组研究和蛋白质组发现研究 样本(项目1和3，以及岩心C和D)。第三，核心将专注于解开 新的趋化因子，HIMF(项目4)，使用多种蛋白质组策略进行最大限度的检测。 免疫沉淀、1D凝胶电泳法或iTRAQTM标记和质谱法将 被用来识别结合伙伴。由于磷酸化在细胞因子信号转导中的突出作用， 将固定化金属亲和层析与液-质联用联用 用于丰富和鉴定各种结合伙伴的特定磷酸化氨基酸残基。 最后，核心将执行计算建模，以开发多环芳烃效应的工作细胞模型 基于蛋白质组、生理和基因组学数据的右室心肌细胞(项目2， 4和5，以及核心C和D)。这项工作将在心血管生物信息学中心进行 和建模，由雷蒙德·L·温斯洛博士执导。

The major goals of Core E are to identify novel biomarkers aimed at the characterization of pulmonary vascular and right ventricle remodeling in severe pulmonary arterial hypertension (PAH) based on state of the art proteomic technology. Core E, with Cores B-D,supports Projects 1,3,4, and 5 of this SCCOR with essential experimental services based on four strategies. First, the Core, using broad-based proteomic analysis, will characterize the changes in lung and right ventricular myocyte proteomes that occur during the development of experimental and human PAH (Projects 1, 3, 4 and 5 and Cores C and D). This strategy will be supported by the existing proteomic infrastructure and established expertise within the JHU- NHLBI Proteomics Center and the JHU Bayview Proteomics Center. In-depth analysis using multiple proteomic platforms, including two-dimensional differential imaging electrophoresis, two-dimensional liquid chromatography, O16/O18 isotopic labeling, isobaric tag for relative and absolute and quantification (iTRAQTM), MALDI-TOF and liquid chromatography/mass spectrometry will be used to maximize proteome coverage, while allowing characterization of protein isoforms and post-translational modifications. Second, the Core will provide a centralized facility to analyze, compile and correlate quantitative levels of candidate biomarkers, including novel markers arising from the discovery platforms as well as additional potentially relevant candidate biomarkers (e.g.VEGF, HIMF, MMP-9 and Granzyme B) in scleroderma-relatedPAH. Beckman A2 multiplex immunoassay will be used to evaluate serum samples while the chemical InkJet printer (CHIP, Proteome Systems) will be used for our targeted approach to confirm expression differences deduced from genomic studies and proteomic discovery studies using lung and right ventricle tissue samples (Projects 1 and 3, and Cores C and D). Third, the Core will focus on unraveling the interactome of the novel chemokine, HIMF (Project 4), using multiple proteomic strategies for maximal detection. Immunoprecipitation followed by 1D gel electrophoresis or iTRAQTM labeling and mass spectrometry will be used to identify binding partners. Due to the prominent role of phosphorylation in cytokine signaling, immobilized metal affinity chromatography online with liquid chromatography/mass spectrometry will be used to enrich and identify the specific phosphorylated amino acid residues of the various binding partners. Finally, the Core will perform computational modeling to develop a working cellular model of PAH effects on the right ventricular cardiac myocyte based on the proteomic, physiological and genomics data (Projects 2, 4 and 5, and Cores C and D). This work will be performed at the Center for Cardiovascular Bioinformatics and Modeling, directed by Raimond L. Winslow, PhD.