Top Down Proteomics Of Myofilaments In Heart Failure

描述（由申请人提供）：心力衰竭是美国男性和女性死亡的主要原因。心衰的潜在分子和细胞机制是非常复杂和知之甚少。关键肌丝调节蛋白（KMRPs），包括心肌肌钙蛋白（cTn）、原肌球蛋白（Tm）、肌球蛋白调节轻链2 （RLC2）和心肌肌球蛋白结合蛋白C (cMyBP-C)，在心脏收缩性中起重要作用。假设是外在和内在的压力都触发了分子信号传导过程，导致KMRPs的改变，导致收缩功能障碍，最终导致心力衰竭。最近的研究令人信服地表明，cTnI和cMyBP-C的改变与心功能障碍直接相关。对KMRPs进行无偏见和系统的分析，以全面检测蛋白质修饰的变化，确定哪些位点被修饰或改变，并阐明这些改变在向心力衰竭过渡期间是如何协同作用的，这对于理解潜在的分子机制至关重要。然而，这仍然是一个重大挑战。为了解决这一挑战，我们建议建立一个简单而强大的基于自上而下质谱（MS）的疾病蛋白质组学平台来检测从正常和病变组织中提取的KMRPs，以建立KMRPs改变修饰与心功能障碍之间的相关性。自上而下的质谱法直接分析完整的蛋白质，提供“鸟瞰图”，在一个光谱中同时观察所有可能的修饰，这比传统的自下而上方法测量蛋白质水解消化的肽要可靠得多。集成的自上而下的蛋白质组学平台将提供一个全面的工具，有效地分离从心肌组织中提取的完整的KMRPs，全球检测所有反映外在和内在压力的修饰，3)识别和量化（新的）修饰，并确定在过渡到终末期心力衰竭过程中KMRPs的多种协同改变和ptm在多个靶向位点的分布变化。具体目标包括：1)建立一种自上而下的疾病蛋白质组学技术，高效、灵敏、简便地分离和表征完整的KMRPs。2)测定肥厚和衰竭猪心肌中KMRPs蛋白修饰的改变。3)测定蛋白激酶A （PKA）和蛋白激酶A （PKC）介导的磷酸化对正常猪和病猪心肌KMRPs的功能影响。4)确定一种新的KMRPs改变的功能后果，例如cTn，在调节心脏收缩力方面。该研究项目整合了蛋白质组学和功能研究，其成功将提供正常和患病条件下KMRPs蛋白修饰的全球图谱，并为心力衰竭中收缩功能障碍的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Heart failure is the leading cause of death for both men and women in the United States. The underlying molecular and cellular mechanisms of heart failure are very complex and poorly understood. Key Myofilament Regulatory Proteins (KMRPs), which include cardiac troponin (cTn), tropomyosin (Tm), myosin regulatory light chain 2 (RLC2) and cardiac myosin binding protein C (cMyBP-C), play essential roles in cardiac contractility. The hypothesis is that both extrinsic and intrinsic stresses trigger the molecular signaling processes that result in altered modifications to KMRPs leading to contractile dysfunction and eventually heart failure. Recent studies show convincingly that altered modifications in cTnI and cMyBP-C are directly linked to cardiac dysfunction. An unbiased and systematic analysis of the KMRPs to globally detect the changes in protein modifications, identify which sites are modified or altered, and elucidate how these alterations act in concert during the transition to the heart failure is of paramount importance for the understanding of the underlying molecular mechanisms. However, this remains a major challenge. To address this challenge, we propose to establish a simple and robust top-down mass spectrometry (MS)-based disease proteomics platform to examine KMRPs extracted from both normal and diseased tissues to establish a correlation between altered modifications of KMRPs and cardiac dysfunction. Top-down MS directly analyzes intact proteins providing a "bird's eye view" to observe all possible modifications simultaneously in one spectrum, which is much more reliable than measuring the proteolytically-digested peptides in the conventional bottom-up approach. The integrated top-down proteomics platform will provide a comprehensive tool to effectively separate the intact KMRPs extracted from myocardial tissues, globally detect all modifications that reflect extrinsic and intrinsic stresses, 3) identify and quantify (novel) modifications, and identify multiple concerted alterations in KMRPs and the changes in the distribution of PTMs among multiple targeted sites during the transition to the end- stage heart failure. The specific aims include: 1) Establish an integrated top-down disease proteomics technology for the separation and characterization of intact KMRPs with high efficiency, sensitivity and simplicity. 2) Determine altered protein modifications in KMRPs from hypertrophied and failing swine myocardium. 3) Determine the functional effects of protein kinase A (PKA) and protein kinase A (PKC)- mediated phosphorylation in KMRPs of normal and diseased swine myocardium. 4) Determine the functional consequence of one novel alteration in KMRPs, e.g. cTn, in regulating cardiac contractility. The success of this research project, which integrates proteomics and functional studies, will provide a global map of protein modifications occurring to the KMRPs under normal and diseased conditions and shed new insights into the molecular mechanism of contractile dysfunction in heart failure.