| Coronary heart disease (CHD), including a variety of different pathologies, is the number one cause of death in the United States, and diagnostic tools are required that allow fast, cost-efficient and reliable triage. Recently, cardiac troponins (cTnI and cTnT) have been recommended as new gold standard for diagnosing acute myocardial infarction (AMI). Even though they have higher cardiac specificity than their predecessors, assays for cardiac troponins are still hampered by disease-induced modifications to the analyte upon ischemia/reperfusion injury to the myocardium. Such modifications not only affect troponin detection by a given assay, but also make it very difficult to standardize the numerous assays currently marketed.; The work presented in this thesis was set to develop protocols for the identification of the various forms of cTnI and cTnT that exist in the circulation of patients with CHD. In order to achieve this goal proteomic tools were employed to separate, detect and characterize cardiac troponins in both tissue and serum. A Western blot-direct serum analysis technique was developed to show that cTnI and cTnT undergo substantial degradation during AMI and by-pass surgery, and that modified troponins are detectable in serum of such patients. This was the first direct evidence of disease-induced troponin degradation in humans. The optimization of cTnT separation by 2-dimensional gel electrophoresis demonstrated the challenges cardiac troponins present to proteomic analysis. To overcome limitations of gel electrophoresis, we combined affinity chromatography with mass spectrometry to investigate cardiac troponins in AMI patient serum and presented first direct evidence of a cTnI-C complex in such patients. Based on this work and in collaboration with us, Ciphergen Inc. developed the first experimental cTnI diagnostic assay using ProteinChip®/mass spectrometry. Finally, applying the aforementioned techniques, we started to evaluate skeletal TnI isoforms as diagnostic markers for skeletal muscle disease.; Identification of the exact forms of marker proteins should be the first step in the development of diagnostic assays to guarantee optimal detection of the analyte. We believe that the presented work will assist in optimization of existing troponin assays as well as in the development of new assays for both cardiac and skeletal diseases. |
