Multiple reaction monitoring to identify site-specific troponin I phosphorylated residues in the failing human heart Journal Article

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Authors: Zhang, P; Kirk, J. A.; Ji, W.; dos Remedios, C. G.; Kass, D. A.; Van Eyk, J. E.; Murphy, A. M.
Article 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. METHODS AND 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.
Journal Title: Circulation
Volume: 126
Issue: 15
ISSN: 1524-4539; 0009-7322
Publisher: Unknown  
Journal Place: United States
Date Published: 2012
Start Page: 1828
End Page: 1837
Language: eng
DOI/URL:

10.1161/CIRCULATIONAHA.112.096388
Notes: LR: 20141105; GR: HHSN268201000032C/PHS HHS/United States; GR: HV-10-05(2)/HV/NHLBI NIH HHS/United States; GR: P01HL081427/HL/NHLBI NIH HHS/United States; GR: P01HL77189-01/HL/NHLBI NIH HHS/United States; GR: R01 HL063038/HL/NHLBI NIH HHS/United States; GR: R01 HL63038/HL/NHLBI NIH HHS/United States; JID: 0147763; 0 (Amino Acids); 0 (Troponin I); CIN: Circulation. 2012 Oct 9;126(15):1803-7. PMID: 22972901; NIHMS486876; OID: NLM: NIHMS486876; OID: NLM: PMC3733556; 2012/09/12 [aheadofprint]; ppublish