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Molecular & Cellular Proteomics 7:1838-1849, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry^*,S

Vlad Zabrouskov, Ying Ge^,¶, Jae Schwartz and Jeffery W. Walker^,||

From Thermo Fisher Scientific, San Jose, California 95134 and the Human Proteomics Program and Department of Physiology, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706

Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser²² and a novel site at Ser⁷⁶/Thr⁷⁷, each with partial occupancy (Ser²²: 53%; Ser⁷⁶/Thr⁷⁷: 36%). Top down MS³ analysis of diphosphorylated cTnI revealed occupancy of Ser²³ only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser^22/23 pair. Top down MS of cTnI provides unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing establishment of the relevance of such modifications to physiological functions and disease status.

^|| To whom correspondence may be addressed. Tel.: 608-262-6941; Fax: 605-265-5512; E-mail: jwalker{at}physiology.wisc.edu

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