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Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state
Open accessAbstract
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 [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).”
From PP2A-B′ holoenzyme substrate recognition, regulation and role in cytokinesis (2017) · cites this paper as [40]
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Metadata
- 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
- Publisher
- National Academy of Sciences
- Published
- 2009-08-04
- Volume
- 106
- Issue
- 31
- Pages
- 12658-12663
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC2722289
- 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
- green
- www.ncbi.nlm.nih.gov
- 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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