In Vivo Phosphorylation Site Mapping in Mouse Cardiac Troponin I by High Resolution Top-Down Electron Capture Dissociation Mass Spectrometry: Ser22/23 Are the Only Sites Basally Phosphorylated

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Ayaz-Guner Ş., Zhang J., Li L., Walker J. W., Ge Y.

BIOCHEMISTRY, vol.48, no.34, pp.8161-8170, 2009 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 48 Issue: 34
  • Publication Date: 2009
  • Doi Number: 10.1021/bi900739f
  • Journal Name: BIOCHEMISTRY
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.8161-8170
  • Abdullah Gül University Affiliated: No


Cardiac troponin I (cTnI) is the inhibitory Subunit of cardiac troponin, a key myofilament regulatory protein complex located on the thin filaments of the contractile apparatus. cTnI is uniquely specific for the heart and is widely used in clinics as a serum biomarker for cardiac injury. Phosphorylation of cTnI plays a critical role in modulating cardiac function. cTnI is known to be regulated by protein kinase A and protein kinase C at five sites, Ser22/Ser23, Ser42/44., and Thr143, primarily based on results from in vitro phosphorylation assays by the specific kinase(s). However, a comprehensive characterization of phosphorylation of mouse cTnI occurring in vivo has been lacking. Herein, we have employed top-down mass spectrometry (MS) methodology with electron capture dissociation for precise mapping of in vivo phosphorylation sites of cTnI affinity purified from wild-type and transgenic mouse hearts, As demonstrated, top-down MS (analysis of intact proteins) is an extremely valuable technology for global characterization of labile phosphorylation occurring in vivo without a priori knowledge. Our top-down MS data unambiguously identified Ser22/23 as the only two sites basally phosphorylated in wild-type mouse cTnI with full sequence coverage, which was confirmed by the lack of phosphorylation in cTnI-Ala(2) transgenic mice where Ser22/23 in cTnI have been rendered nonphosphorylatable by mutation to alanine.