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Tropomyosins (Tm) constitute a family of ubiquitous and highly conserved actin-binding proteins, playing essential roles in a variety of biological processes. Tm isoforms produced by multiple Tm encoding genes and alternatively expressed exons along with post-translational modifications (PTMs) regulate Tm function. Therefore, to gain a better understanding of the functional role of Tm, it is essential to fully characterize Tm isoforms. Herein, we developed a top-down high-resolution mass spectrometry (MS) based targeted proteomics method for comprehensive characterization of Tm isoforms. α–Tm was identified to be the predominant isoform in swine cardiac muscle. We further characterized its sequence and localized the PTMs such as acetylation and phosphorylation as well as amino acid polymorphisms. Interestingly, we discovered a “novel” Tm isoform that does not match with any of the currently available swine Tm sequences. A deep sequencing of this isoform by top-down MS revealed an exact match with mouse β–Tm sequence, suggesting that this “novel” isoform is swine β–Tm which is 100% conserved between swine and mouse. Taken together, we demonstrated that top-down targeted proteomics provides a powerful tool for deep sequencing of Tm isoforms from genetic variations together with complete mapping of the PTM sites.

The rapid increase in the prevalence of chronic heart failure (CHF) worldwide underscores an urgent need to identify biomarkers for the early detection of CHF. Post-translational modifications (PTMs) are associated with many critical signaling events during disease progression and thus offer a plethora of candidate biomarkers. We have employed a top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We systematically analyzed 36 clinical human heart tissue samples and identified phosphorylation of cardiac troponin I (cTnI) as a candidate biomarker for CHF. The relative percentages of the total phosphorylated cTnI forms over the entire cTnI populations (%P(total)) were 56.4 ± 3.5%, 36.9 ± 1.6%, 6.1 ± 2.4%, and 1.0 ± 0.6% for postmortem hearts with normal cardiac function (n = 7), early stage of mild hypertrophy (n = 5), severe hypertrophy/dilation (n = 4), and end-stage CHF (n = 6), respectively. In fresh transplant samples, the %P(total) of cTnI from nonfailing donor (n = 4), and end-stage failing hearts (n = 10) were 49.5 ± 5.9% and 18.8 ± 2.9%, respectively. Top-down MS with electron capture dissociation unequivocally localized the altered phosphorylation sites to Ser22/23 and determined the order of phosphorylation/dephosphorylation. This study represents the first clinical application of top-down MS-based quantitative proteomics for biomarker discovery from tissues, highlighting the potential of PTMs as disease biomarkers.