Cardiac troponin T (cTnT) regulates the Ca²⁺-mediated interaction between myosin thick filaments and actin thin filaments during cardiac contraction and relaxation. cTnT is released into the blood following injury, and increased serum levels of the protein are used clinically as a biomarker for myocardial infarction. Moreover, mutations in cTnT are causative in a number of familial cardiomyopathies. With the increasing use of large animal (swine) model to recapitulate human diseases, it is essential to characterize species-dependent protein sequence variants, alternative RNA splicing, and post-translational modifications (PTMs), but challenges remain due to the incomplete database and lack of validation of the predicted splicing isoforms. Herein, we integrated top-down mass spectrometry (MS) with online liquid chromatography (LC) and immunoaffinity purification to comprehensively characterize miniature swine cTnT proteoforms, including those arising from alternative RNA splicing and PTMs. A total of seven alternative splicing isoforms of cTnT were identified by LC/MS from swine left ventricular tissue, with each isoform containing un-phosphorylated and mono-phosphorylated proteoforms. The phosphorylation site was localized to Ser1 for the mono-phosphorylated proteoforms of cTnT1, 3, 4, and 6 by online MS/MS combining collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). Offline MS/MS on Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer with CAD and electron capture dissociation (ECD) was then utilized to achieve deep sequencing of mono-phosphorylated cTnT1 (35.2 kDa) with a high sequence coverage of 87%. Taken together, this study demonstrated the unique advantage of top-down MS in the comprehensive characterization of protein alternative splicing isoforms together with PTMs.

中文翻译：

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通过自上而下的质谱法全面鉴定猪心脏肌钙蛋白T蛋白形式

心肌肌钙蛋白T（c TnT）调节心肌收缩和舒张过程中肌球蛋白粗丝和肌动蛋白细丝之间的Ca ²⁺介导的相互作用。c TnT在损伤后释放到血液中，并且该蛋白的血清水平升高在临床上被用作心肌梗塞的生物标志物。而且，c中的突变TnT是许多家族性心肌病的病因。随着大型动物（猪）模型在人类疾病概述中的应用日益增多，表征物种依赖性蛋白序列变体，替代性RNA剪接和翻译后修饰（PTM）至关重要，但由于数据库和数据库的不完整，挑战依然存在。缺乏对预期剪接同工型的验证。本文中，我们将自上而下的质谱（MS）与在线液相色谱（LC）和免疫亲和纯化相结合，以全面表征微型猪c TnT蛋白形式，包括那些由其他RNA剪接和PTM产生的蛋白形式。共有7种c的可变剪接亚型通过LC / MS从猪左心室组织中鉴定出TnT，每种同工型均含有未磷酸化和单磷酸化的蛋白形式。通过在线MS / MS结合碰撞活化解离（CAD）和电子转移解离（ETD），将c TnT1、3、4和6的单磷酸化蛋白形式的磷酸化位点定位于Ser1 。然后使用带有CAD和电子俘获解离（ECD）的傅立叶变换离子回旋共振（FT-ICR）质谱仪上的离线MS / MS进行单磷酸化c的深度测序TnT1（35.2 kDa）具有87％的高序列覆盖率。综上所述，这项研究证明了自上而下的MS在蛋白质替代剪接同工型与PTM一起全面表征中的独特优势。