Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T.
Phosphorylation, but not alternative splicing or proteolytic degradation, is conserved in human and mouse cardiac troponin T.
复制标题
人和小鼠心肌肌钙蛋白 T 中磷酸化是保守的，但选择性剪接或蛋白水解降解不保守。
作者：
Zhang,Jiang;Zhang,Han;Ayaz-Guner,Serife;Chen,Yi-Chen;Dong,Xintong;Xu,Qingge;Ge,Ying
心肌肌钙蛋白T（cTnT）是肌钙蛋白复合物的原肌球蛋白结合亚基，在Ca ~（2+）介导的肌动蛋白细丝和肌球蛋白粗丝之间的相互作用中起着关键的调节作用。心肌肌钙蛋白T的翻译后修饰（PTM）和选择性剪接可能是心肌收缩力的重要调节机制。然而，在cTnT存在于体内的PTM和选择性剪接亚型的完整表征是缺乏的。自上而下的蛋白质质谱（MS）分析整个蛋白质，从而提供了所有类型的修饰，包括PTM和序列变异，同时在一个光谱中，没有优先知识的全局视图。在这项研究中，我们应用了一个集成的免疫亲和色谱和自上而下的MS方法，全面表征PTM和选择性剪接的cTnT异构体从健康的人类和野生型小鼠心脏组织纯化。高分辨率傅立叶变换MS显示，人cTnT（hcTnT）和小鼠cTnT（mcTnT）具有相似的磷酸化模式，而mcTnT比hcTnT观察到更高的分子异质性。通过电子捕获解离和碰撞活化解离对单磷酸化hcTnT和mcTnT进行进一步的MS/MS片段化，明确鉴定了Ser 1为保守的体内磷酸化位点。相比之下，我们确定了一个单一的剪接hcTnT的亚型，但三个选择性剪接mcTnT的亚型。此外，我们观察到不同的hcTnT和mcTnT的蛋白水解降解产物。本研究还证明了自上而下MS/MS与互补裂解技术的优势，用于鉴定cTnT高酸性N-末端区域的修饰位点。
Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca2+-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum withouta prioriknowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.