title: Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation state.
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.
authors.affiliation: Human Proteomics Program and Department of Physiology, School of Medicine and Public Health, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706, USA. yge@physiology.wisc.edu
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
authors.forename: Inna N
authors.initials: IN
authors.lastname: Rybakova
authors.forename: Qingge
authors.initials: Q
authors.lastname: Xu
authors.forename: Richard L
authors.initials: RL
authors.lastname: Moss
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D011994:Recombinant Proteins; D000099163:Myosin Binding Protein C