| First Author | Ge Y | Year | 2009 |
| Journal | Proc Natl Acad Sci U S A | Volume | 106 |
| Issue | 31 | Pages | 12658-63 |
| PubMed ID | 19541641 | Mgi Jnum | J:151897 |
| Mgi Id | MGI:4355493 | Doi | 10.1073/pnas.0813369106 |
| Citation | Ge Y, et al. (2009) Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state. Proc Natl Acad Sci U S A 106(31):12658-63 |
| abstractText | 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. |
