Proteomic approaches to validate novel cardiac biomarkers for myocardial infarcti

说明(申请人提供)：心肌肌球蛋白结合蛋白-C(cMyBP-C)的磷酸化可调节肌节结构和心肌收缩能力，并提供心脏保护作用。我的长期目标是确定cMyBP-C磷酸化在收缩功能中的作用(S)，以便了解心脏保护的分子机制。我们最近发现：(1)cMyBP-C是一种易于释放的可溶性肌丝；(2)cMyBP-C的去磷酸化导致其降解；(3)cMyBP-C在心肌梗死(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，我们还发现心肌梗死动物模型和患者的血浆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，核实和确认血浆cMyBP-C的准确数量是下一个关键步骤。因此，使用选择性和特定的蛋白质组学方法，短期目标是开发一种准确定量血浆cMyBP-C水平的分析方法。我的中心假设是cMyBP-C是一种有骨性的早期、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，该提案的总体目标如下：确定cMyBP-C特定的氨基酸区域，以便利用LC-MS-MS和选择性反应监测(SRM)方法建立基于蛋白质组的选择性分析(具体目标1)；验证SRM方法用于量化动物和人类血浆样本中cMyBP-C水平的准确性(具体目标2)；以及与传统的夹心ELISA法交叉验证SRM分析(具体目标3)。从缺血-再灌注损伤的小鼠和心肌梗塞患者身上采集的血浆样本将用于这些分析，与幼稚和假手术的小鼠和正常的健康对照组进行比较。对蛋白质组学方法的验证将导致一种能够准确测量循环系统中cMyBP-C水平的分析方法，并将其定义为早期释放的、心脏特异的和选择性的早发性心肌梗死标志物。 公共卫生相关性：这项建议旨在确认心脏结合蛋白-C(cMyBP-C)作为心肌梗死早期循环生物标志物的有效性。具体地说，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，从而开发出促进心脏护理的诊断分析方法。

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.