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.
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.
Ayaz-Guner
Serife
S
Human Proteomics Program and Department of Physiology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Zhang
Jiang
J
Li
Lin
L
Walker
Jeffery W
JW
Ge
Ying
Y
D000595:Amino Acid Sequence; D000818:Animals; D004583:Electrons; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012680:Sensitivity and Specificity; D012694:Serine; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019210:Troponin I; D012694:Serine