Cited by 154Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsCardiomyopathy and Myosin Studies

Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state

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Ge, Ying, Rybakova, Inna N., Xu, Qingge, Moss, Richard L.·2009·Proceedings of the National Academy of Sciences

Abstract

Cardiac myosin binding protein C (cMyBP-C), bound to the sarcomere'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.

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  • High-resolution top-down MS analysis revealed that Cyk4 is presented as mono-, bis-, tris-, tetrakis- and pentakis-phosphorylated proteoforms, and no unphosphorylated Cyk4 is detected.

    “High-resolution top-down MS analysis [39, 40] revealed that Cyk4 is presented as mono-, bis-, tris-, tetrakis- and pentakis-phosphorylated proteoforms. No unphosphorylated CyK4 is detected (Figure 4e, left panel).”

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Authors
Ge, Y., Rybakova, I. N., Xu, Q., & Moss, R. L.
Affiliations
University of Wisconsin–Madison (US)
Venue
Proceedings of the National Academy of Sciences
Published
2009-08-04
Volume
106
Issue
31
Pages
12658-12663
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
OpenAlex
W2048461578
Cited by
154
References
50
Citations by year
2026: 5 · 2025: 1 · 2024: 4 · 2023: 1 · 2022: 2 · 2021: 5 · 2020: 5 · 2019: 14 · 2018: 10 · 2017: 13 · 2016: 9 · 2015: 8 · 2014: 16 · 2013: 13 · 2012: 14
FWCI
6.87
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
National Heart, Lung, and Blood Institute (R37HL082900, R37 HL82900)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cardiomyopathy and Myosin Studies
Keywords
myosin binding protein C, cMyBP-C, protein phosphorylation, phosphorylation site mapping, protein truncation, top-down mass spectrometry, electron capture dissociation, middle-down mass spectrometry, limited proteolysis, post-translational modification
MeSH terms
Myosin Binding Protein C, Amino Acid Sequence, Carrier Proteins, Molecular Sequence Data, Phosphorylation, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry

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