Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.
Top-down proteomics reveals concerted reductions in myofilament and Z-disc protein phosphorylation after acute myocardial infarction.
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DOI：
10.1074/mcp.m114.040675
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发表时间：
2014-10
期刊：
影响因子：
--
通讯作者：
Ge Y
中科院分区：
文献类型：
--
作者：
Peng Y;Gregorich ZR;Valeja SG;Zhang H;Cai W;Chen YC;Guner H;Chen AJ;Schwahn DJ;Hacker TA;Liu X;Ge Y
心力衰竭（HF）是世界范围内发病率和死亡率的主要原因，最常见的是心肌梗死。然而，心肌梗死后立即驱动心功能不全的分子变化仍然知之甚少。肌丝蛋白在心力衰竭的信号接收和转导中起着关键作用，负责心脏的收缩和舒张。肌丝蛋白的翻译后修饰提供了心脏功能搏动调节的机制。因此，它是至关重要的，以获得一个全面的了解，在梗死后心肌的早期分子事件的调节所涉及的肌丝蛋白的翻译后修饰。我们已经开发了一种新的液相色谱-质谱为基础的自上而下的蛋白质组学策略，以全面评估从最小量的心肌组织中提取的肌丝亚蛋白质组中的关键心脏蛋白质的修饰，具有高重现性和高通量。整个过程，包括组织均质化，肌丝提取和在线LC/MS，需要不到三个小时。值得注意的是，通过这种新的自上而下的蛋白质组学技术，我们发现了急性梗死猪心肌中三种关键心脏蛋白磷酸化的协同显著降低：心肌肌钙蛋白I和肌丝的肌球蛋白调节轻链，以及出乎意料的Z盘的Enigma同源异构体2（ENH 2）。此外，自上而下的MS使我们能够全面测序这些蛋白质并确定其磷酸化位点。这是第一次，我们已经确定了ENH 2的序列，并确定其为磷蛋白。ENH 2定位于Z盘，其作为心脏信号传导中的节点的作用已越来越被认识到。因此，我们的蛋白质组学发现开辟了新的途径，研究肌丝和Z盘之间的协调信号在早期分子事件，有助于心功能障碍和进展为HF。
Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.