Quantitative Proteomic Analysis of the Intact Human Heart

描述(由申请人提供)：此R21申请(最多10页)是响应PA-03-015“NHLBI创新研究资助计划”而提交的，该计划为初步研究提供支持，旨在证明利用现有的人类生物标本收集的新方法的可行性。我们建议分析一种现有的生化组分，它是NHLBI资助的题为“心肌衰竭中的遗传-肾上腺素能相互作用”的研究(PI-Michael Bristow)中描述的样品制备策略的副产品。基金RO1的总体设计策略是比较来自非衰竭对照受试者和特发性扩张型心肌病(IDCM)双室衰竭受试者的完整心脏样本之间的基因表达，以及对IDCM受试者进行纵向、连续抽样研究，比较治疗和β-受体阻滞剂的治疗，以确定哪种肾上腺素能受体途径对衰竭的重塑心脏的基因表达变化负责。该R21提案的总体目标是通过一项平行的蛋白质组学研究来增强微阵列分析。我们建议开发一种定量蛋白质组学策略来分析从RNA提取的活组织切片大小的人心脏样本中蛋白质水平的变化，并确定完整衰竭心脏对β-受体阻滞剂的反应中蛋白质表达的时间变化。在具体目标1中，将使用多维蛋白质识别技术(MudPIT)从对照受试者和诊断为IDCM的受试者的样本中识别蛋白质及其修饰。在具体目标2中，将使用定量蛋白质组学从患者在β-受体阻滞剂治疗过程中收集的系列样本中监测选定蛋白质及其修饰的差异表达。这一蛋白质组学研究将进一步加深我们对心力衰竭分子机制的理解。通过开发分析先前存在的心脏活检样本的方法，人类研究的冗余性将显著减少。

DESCRIPTION (provided by applicant): This R21 application (10 pages max) is being submitted in response to PA-03-015 "NHLBI Innovative Research Grant Program" which provides support for preliminary studies designed to demonstrate the feasibility of novel approaches using existing collections of human biological specimens. We propose to analyze an existing biochemical fraction that is a by-product of the sample preparation strategy described in the NHLBI-funded study (Pi-Michael Bristow) entitled "Genetic- Adrenergic Interactions in Myocardial Failure." The overall design strategy for the funded RO1 is to compare the gene expression between intact heart samples from nonfailing control subjects and subjects with biventricular failure from idiopathic dilated cardiomyopathy (IDCM), as well as a longitudinal, serial sampling study in IDCM subjects comparing treatments with beta-blockade to determine which adrenergic receptor pathway is responsible for changes in gene expression in the failing remodeled heart. The overall objective of this R21 proposal is to augment the microarray analysis with a parallel proteomic study. We propose to develop a quantitative proteomic strategy to analyze changes in protein levels from RNA-extracted biopsy- sized human heart samples and to determine the temporal changes in protein expression in the intact failing heart in response to beta-blockade. In Specific Aim 1, proteins and their modifications will be identified from samples taken from control subjects and subjects diagnosed with IDCM using Multi-dimensional Protein Identification Technology (MudPIT). In Specific Aim 2, differential expression of selected proteins and their modifications will be monitored from serial samples collected from patients during the course of beta- blockade treatment using quantitative proteomics. This proteomic study will further our understanding of the molecular mechanisms contributing to heart failure. By developing methods to analyze previously existing heart biopsy specimens, the redundancy of human studies will be significantly reduced.