1. Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state
- Author
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Richard L. Moss, Ying Ge, Inna N. Rybakova, and Qingge Xu
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Multidisciplinary ,biology ,Chemistry ,Binding protein ,Molecular Sequence Data ,Tandem mass spectrometry ,Recombinant Proteins ,Biochemistry ,Tandem Mass Spectrometry ,Physical Sciences ,Myosin ,biology.protein ,Biophysics ,Phosphorylation ,Titin ,Protein phosphorylation ,Amino Acid Sequence ,Carrier Proteins ,Peptide sequence ,Actin - 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 (
- Published
- 2009