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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state
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Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

机译：心肌肌球蛋白结合蛋白C的自上而下的高分辨率质谱表明截短改变了蛋白的磷酸化状态

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摘要

Cardiac myosin binding protein C (cMyBP-C), bound to the sar-comere's myosin thick filament, plays an important role in the regulation of muscle contraction. cMyBP-C is a large multidomain protein that interacts with myosin, titin, and possibly actin. Mutations in cMyBP-C are the most common known cause of heritable hypertrophic cardiomypathies. Phosphorylation of cMyBP-C plays an essential role in the normal cardiac function. cMyBP-C (142 kDa) has 81 serine and 73 threonine residues presenting a major challenge for unequivocal identification of specific phosphorylation sites. Top-down mass spectrometry, which directly analyzes intact proteins, is a powerful technique to universally observe and quantify protein posttranslational modifications without a priori knowledge. Here, we have extended top-down electron capture dissociation mass spectrometry to comprehensively characterize mouse cMyBP-C expressed in baculovirus. We have unambiguously identified all of the phosphorylation sites in the truncated (28-115 kDa) and full-length forms of cMyBP-C (142 kDa) and characterized the sequential phosphorylations, using a combination of top-down and middle-down (limited proteolysis) MS approach, which ensures full sequence coverage. Unit mass resolution and high mass accuracy (<5 ppm) have been achieved for a 115-kDa protein (the largest protein isotopically resolved to date). Remarkably, we discovered that truncations in recombinant proteins, even a seemingly minor one, can dramatically alter its phosphorylation state, which is significant because truncated recombinant proteins are routinely substituted for their full-length forms in crystal structure and functional studies. Our study provides direct evidence of alterations in the posttranslational state between the truncated and full-length recombinant proteins, which can lead to variations in structure and function.
机译：心肌肌球蛋白结合蛋白C（cMyBP-C）与sar-comere的肌球蛋白粗丝结合，在调节肌肉收缩中起重要作用。 cMyBP-C是一种大型的多域蛋白，可与肌球蛋白，肌动蛋白和肌动蛋白相互作用。 cMyBP-C突变是遗传性肥厚型心肌病的最常见已知原因。 cMyBP-C的磷酸化在正常心脏功能中起重要作用。 cMyBP-C（142 kDa）具有81个丝氨酸和73个苏氨酸残基，这对明确鉴定特定的磷酸化位点提出了重大挑战。自上而下的质谱法直接分析完整的蛋白质，是一种无需先验知识即可普遍观察和量化蛋白质翻译后修饰的强大技术。在这里，我们已经扩展了自上而下的电子捕获解离质谱，以全面表征杆状病毒中表达的小鼠cMyBP-C。我们已明确鉴定出截短的（28-115 kDa）和全长形式的cMyBP-C（142 kDa）的所有磷酸化位点，并结合了自上而下和中-下的特点对连续的磷酸化进行了表征（有限蛋白水解）MS方法，可确保完整序列覆盖。对于115 kDa的蛋白质（迄今为止最大的同位素同位素分解蛋白质），已经实现了单位质量分辨率和高质量准确度（<5 ppm）。值得注意的是，我们发现重组蛋白中的截短，即使是看似很小的蛋白，也可以显着改变其磷酸化状态，这很重要，因为在晶体结构和功能研究中，截短的重组蛋白通常被其全长形式取代。我们的研究提供了截断和全长重组蛋白之间翻译后状态变化的直接证据，这可能导致结构和功能的变化。

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  • 作者

    Ying Ge; Inna N. Rybakova; Qingge Xu; Richard L. Moss;

  • 作者单位

    Human Proteomics Program, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706 Department of Physiology, School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706;

    Department of Physiology, School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706;

    Human Proteomics Program, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706 Department of Physiology, School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706;

    Human Proteomics Program, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706 Department of Physiology, School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    electron capture dissociation; hypertrophic cardiomypathy;

    机译：电子俘获解离;肥厚型心肌病;

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