High Resolution Top-Down MS/MS Reveals Single Amino Acid Sequence Polymorphisms in Rat Cardiac Troponin

Abstract

Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex. Two of the three subunits, cTnI and cTnT, are expressed only in cardiac muscle and are widely used in the clinic as serum biomarkers of cardiac injury. cTnI and cTnT are subject to extensive post-translational modification such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. In order to obtain a global view of the state of post-translational modification of cTn, we are performing high resolution top-down mass spectrometry on cTn subunits isolated from native tissues. Whole cTn complexes affinity purified from a single rat heart were analyzed in a 7 Tesla Thermo LTQ-FT-ICR mass spectrometer equipped with an ESI source. High resolution MS spectra of cTn from healthy adult rats showed molecular ions for intact cTnT and cTnI as well as phosphorylation and acetylation patterns similar to human cTnI (Zabrouskov et al., 2008 Mol Cell Proteomics, in press). ‘Shadow peaks’ of similar intensity to parent peaks were detected exhibiting masses of cTnI + 16 Da and cTnT + 128 Da, suggestive of single amino acid polymorphisms. Tandem mass spectrometry (MS/MS) analysis by ECD and CAD fragmentation of intact and protease-digested cTn subunits localized an Ala/Ser polymorphism at residue 7 of cTnI, and an additional Gln within a 3 residue alternative splice site beginning at residue 192 of cTnT. High resolution top-down MS/MS has revealed intriguing heterogeneity not only in the extent of phosphorylation, but also in amino acid sequences of cTnI and cTnT even within a single rat heart. Supported by NIH, AHA, UW-CVRC & Wisconsin Partnership for a Healthy Future.