Proteomic approaches to validate novel cardiac biomarkers for myocardial infarcti

验证心肌梗塞新型心脏生物标志物的蛋白质组学方法

• 批准号: 8705576
• 负责人: Sakthivel Sadayappan
• 金额: $ 9.17万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8705576
• 关键词: Amino Acid Sequence; Amino Acids; Animal Model; Animals; Appearance; Binding Proteins; Biological Assay; Biological Markers; Blood; Cardiac; Cardiac Myocytes; Cardiac Myosins; Cardiovascular system; Caring; Cleaved cell; Creatine Kinase; Data; Development; Diagnosis; Diagnostic; Early Diagnosis; Enzyme-Linked Immunosorbent Assay; Genes; Goals; Gold; Half-Life; Heart; Heart failure; Human; Ischemia; Isotonic Exercise; Kinetics; Label; Laboratories; Liquid Chromatography; Mass Spectrum Analysis; Measurable; Measures; Methods; Microfilaments; Molecular; Monitor; Mus; Mutation; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myoglobin; N-terminal; Pathogenesis; Patients; Peptides; Phosphorylation; Pilot Projects; Plasma; Protein C; Protein Dephosphorylation; Proteins; Proteolysis; Proteomics; Reaction; Reperfusion Injury; Reperfusion Therapy; Resistance; Role; Sampling; Sarcomeres; Signal Transduction; Specificity; Staging; Structure; Testing; Thick Filament; Thin Filament; Troponin I; base; bone; early onset; genetic regulatory protein; injured; myosin-binding protein C; novel; tandem mass spectrometry; time use; verification and validation

DESCRIPTION (provided by applicant): Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates sarcomeric structure, as well as myocardial contractility, and confers cardioprotection. My long-term goal is to define the role(s) of cMyBP-C phosphorylation in contractile function in order to understand the molecular mechanisms that underlie cardioprotection. We recently showed that (1) cMyBP-C is an easily releasable and soluble myofilament, (2) dephosphorylation of cMyBP-C results in its degradation and (3) release into the blood post-myocardial infarction (MI) and (4) its N'-fragments appear within 30 minutes of ischemia-reperfusion injury. In addition, we showed that plasma cMyBP-C levels are significantly increased in animal models and patients with MI. Strikingly, the level of plasma cMyBP-C is significantly higher than the gold standard plasma cardiac troponin I (2.0-fold molar). However, verification and validation of the precise amount of plasma cMyBP-C is the next critical step. Therefore, using selective and specific proteomic approaches, the short-term goal is to develop an assay that precisely quantitates the levels of plasma cMyBP-C. My central hypothesis is that cMyBP-C is a bone fide early, selective and measurable cardiac-specific biomarker, which appears within 30 minutes of ischemia. Thus, the overall objectives of the proposal are as follows: determine a cMyBP-C-specific amino acid region in order to develop a selective proteomics-based assay using the liquid chromatography-tandem-mass spectrometry (LC-MS-MS) and selective reaction monitoring (SRM) approaches (Specific Aim 1); verify the accuracy of the SRM approach for quantifying plasma cMyBP-C levels in both animal and human plasma samples (Specific Aim 2); and cross-validate the SRM assay with the conventional sandwich ELISA assay (Specific Aim 3). Plasma samples taken from ischemia- reperfusion-injured mice and patients with MI will be used in these analyses, compared to naive and sham operated mice and normal healthy controls. Validating the proteomic approaches will result in an assay that can accurately measure the level of cMyBP-C in the circulatory system, as well as define its presence as an early-released, cardiac-specific and selective marker of early-onset MI.

描述（由申请人提供）：心肌肌球蛋白结合蛋白c （cMyBP-C）的磷酸化调节肌肉结构，以及心肌收缩性，并赋予心脏保护作用。我的长期目标是确定cMyBP-C磷酸化在收缩功能中的作用，以了解心脏保护的分子机制。我们最近发现(1)cMyBP-C是一种易于释放和溶解的肌丝，(2)cMyBP-C的去磷酸化导致其降解，(3)心肌梗死（MI）后释放到血液中，(4)其N'片段在缺血再灌注损伤后30分钟内出现。此外，我们发现动物模型和心肌梗死患者血浆cMyBP-C水平显著升高。引人注目的是，血浆cMyBP-C水平显著高于金标准血浆心肌肌钙蛋白I（2.0倍摩尔）。然而，验证和确认血浆cMyBP-C的精确量是下一个关键步骤。因此，使用选择性和特异性蛋白质组学方法，短期目标是开发一种精确定量血浆cMyBP-C水平的测定方法。我的主要假设是cMyBP-C是一种早期、选择性和可测量的心脏特异性生物标志物，在缺血30分钟内出现。因此，该提案的总体目标如下：确定cmybp - c特异性氨基酸区域，以便使用液相色谱-串联质谱（LC-MS-MS）和选择性反应监测（SRM）方法开发基于选择性蛋白质组学的分析（Specific Aim 1）；验证SRM方法定量动物和人血浆样品中血浆cMyBP-C水平的准确性（特异性目标2）；并将SRM试验与传统的夹心ELISA试验（Specific Aim 3）交叉验证。这些分析将使用缺血再灌注损伤小鼠和心肌梗死患者的血浆样本，与未手术、假手术小鼠和正常健康对照进行比较。验证蛋白质组学方法将产生一种能够准确测量循环系统中cMyBP-C水平的分析方法，并将其定义为早发性心肌梗死的早期释放、心脏特异性和选择性标记物。