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.
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