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 top-down quantitative proteomics methodology for comprehensive assessment of PTMs in whole proteins extracted from normal and diseased tissues. We have systematically analyzed thirty-six 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 (%Ptotal) 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 %Ptotal of cTnI from non-failing 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 PTM as disease biomarkers.

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Background Cardiac troponin I (cTnI) is the current standard biomarker for diagnosing acute myocardial infarction and for risk-stratification of acute coronary syndromes in patients. However it remains unclear how the epitope specificity of antibodies in immunoassays influences the detection of various modified forms of cTnI. Methods Four mouse anti-human cTnI monoclonal antibodies targeting different regions of human cTnI were chosen for immunoaffinity purification of cTnI from human and swine cardiac tissue. High-resolution intact protein mass spectrometry was employed to assess the comparative performance of these four antibodies in detecting modified forms of cTnI. Results Our data revealed that antibody selection significantly impacts the relative protein yield of cTn from immunoaffinity purification. Remarkably, a single amino acid variation in cTnI (G->S) in the epitope region completely abolished the binding between monoclonal antibody 560 and swine cTnI in solution. Moreover, proteolytic degradation around the epitope region severely compromised the detection of proteolytic fragment forms of cTnI by monoclonal antibodies. In contrast, the phosphorylation status near the epitope region did not significantly affect the antibody recognition of cTnI. Conclusion Caution needs to be taken in the interpretation of the data produced by immuno-assays with monoclonal antibodies against various epitopes of cTnI.