Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, WI, 53705, USA.
Human Proteomics Program, University of Wisconsin-Madison, Madison, WI, 53705, USA.
J Am Soc Mass Spectrom. 2018 Jun;29(6):1284-1294. doi: 10.1007/s13361-018-1925-y. Epub 2018 Apr 9.
Cardiac troponin T (cTnT) regulates the Ca-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. Graphical Abstract ᅟ.
心肌肌钙蛋白 T（cTnT）在心脏收缩和舒张过程中调节肌球蛋白粗丝和肌动蛋白细丝之间的 Ca 介导相互作用。cTnT 在损伤后释放到血液中，并且蛋白质的血清水平升高临床上用作心肌梗死的生物标志物。此外，cTnT 的突变是许多家族性心肌病的原因。随着大型动物（猪）模型越来越多地用于重现人类疾病，对种属依赖性蛋白质序列变体、选择性 RNA 剪接和翻译后修饰（PTM）进行特征描述至关重要，但由于数据库不完整和预测剪接异构体缺乏验证，仍然存在挑战。在此，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，全面表征小型猪 cTnT 蛋白变体，包括那些来自选择性 RNA 剪接和 PTM 的蛋白变体。通过 LC/MS 从猪左心室组织中鉴定出七种 cTnT 的选择性剪接异构体，每个异构体均包含未磷酸化和单磷酸化的蛋白变体。通过在线 MS/MS 结合碰撞诱导解离（CAD）和电子转移解离（ETD），将 cTnT1、3、4 和 6 的单磷酸化蛋白变体的磷酸化位点定位到 Ser1。然后，在线傅立叶变换离子回旋共振（FT-ICR）质谱仪上使用 CAD 和电子俘获解离（ECD）对离线 MS/MS 进行，以实现单磷酸化 cTnT1（35.2 kDa）的深度测序，序列覆盖率高达 87%。总之，这项研究表明，自上而下的 MS 在全面表征蛋白质选择性剪接异构体以及 PTM 方面具有独特的优势。
