Proteomic approaches to validate novel cardiac biomarkers for myocardial infarcti

描述（由申请方提供）：心肌肌球蛋白结合蛋白-C（cMyBP-C）的磷酸化调节肌节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是一种容易释放和可溶的肌丝，（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-再灌注损伤的30分钟内出现。此外，我们发现，在动物模型和MI患者中，血浆cMyBP-C水平显著升高。值得注意的是，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。我的中心假设是，cMyBP-C是一种真正的早期，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，本研究的总体目标如下：确定cMyBP-C的特异性氨基酸区域，以建立基于液相色谱-串联质谱的选择性蛋白质组学检测方法（LC-MS-MS）和选择性反应监测（SRM）方法（具体目标1）;验证SRM方法定量动物和人血浆样本中血浆cMyBP-C水平的准确性（特异性目标2）;并与常规夹心ELISA试验交叉验证SRM试验（特异性目标3）。从缺血-再灌注损伤的小鼠和MI患者采集的血浆样品将用于这些分析，与未处理和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可以准确测量循环系统中cMyBP-C水平的检测方法，并将其定义为早发性MI的早期释放、心脏特异性和选择性标志物。 公共卫生相关性：该提议旨在确认心脏结合蛋白-C（cMyBP-C）（一种肌节心脏特异性粗丝组装蛋白）作为心肌梗死的早期循环生物标志物的功效。具体而言，拟议的研究将核实和 通过先进的蛋白质组学方法验证cMyBP-C血浆水平，从而开发出推进心脏护理的诊断检测方法。

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. PUBLIC HEALTH RELEVANCE: This proposal aims to confirm the efficacy of cardiac binding protein-C (cMyBP-C), a sarcomeric cardiac- specific thick filament assembly protein, as an early circulatory biomarker for myocardial infarction. Specifically, the proposed studies will verify and validate cMyBP-C plasma levels through advanced proteomic approaches, leading to the development of diagnostic assays that advance cardiac care.