title: Multiple reaction monitoring to identify site-specific troponin I phosphorylated residues in the failing human heart.
abstract: BACKGROUND
  Human cardiac troponin I is known to be phosphorylated at multiple amino acid residues by several kinases. Advances in mass spectrometry allow sensitive detection of known and novel phosphorylation sites and measurement of the level of phosphorylation simultaneously at each site in myocardial samples.
  
  
  RESULTS
  On the basis of in silico prediction and liquid chromatography/mass spectrometry data, 14 phosphorylation sites on cardiac troponin I, including 6 novel residues (S4, S5, Y25, T50, T180, S198), were assessed in explanted hearts from end-stage heart failure transplantation patients with ischemic heart disease or idiopathic dilated cardiomyopathy and compared with samples obtained from nonfailing donor hearts (n=10 per group). Thirty mass spectrometry-based multiple reaction monitoring quantitative tryptic peptide assays were developed for each phosphorylatable and corresponding nonphosphorylated site. The results show that in heart failure there is a decrease in the extent of phosphorylation of the known protein kinase A sites (S22, S23) and other newly discovered phosphorylation sites located in the N-terminal extension of cardiac troponin I (S4, S5, Y25), an increase in phosphorylation of the protein kinase C sites (S41, S43, T142), and an increase in phosphorylation of the IT-arm domain residues (S76, T77) and C-terminal domain novel phosphorylation sites of cardiac troponin I (S165, T180, S198). In a canine dyssynchronous heart failure model, enhanced phosphorylation at 3 novel sites was found to decline toward control after resynchronization therapy.
  
  
  CONCLUSIONS
  Selective, functionally significant phosphorylation alterations occurred on individual residues of cardiac troponin I in heart failure, likely reflecting an imbalance in kinase/phosphatase activity. Such changes can be reversed by cardiac resynchronization.
authors.affiliation: Department of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Pingbo
authors.initials: P
authors.lastname: Zhang
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Kirk
authors.forename: Weihua
authors.initials: W
authors.lastname: Ji
authors.forename: Cristobal G
authors.initials: CG
authors.lastname: dos Remedios
authors.forename: David A
authors.initials: DA
authors.lastname: Kass
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
authors.forename: Anne M
authors.initials: AM
authors.lastname: Murphy
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D058406:Cardiac Resynchronization Therapy; D004285:Dogs; D006333:Heart Failure / Q000378:metabolism* / Q000503:physiopathology / Q000628:therapy; D016027:Heart Transplantation; D006801:Humans; D013058:Mass Spectrometry; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D019210:Troponin I / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D019210:Troponin I