Determination of cardiac troponin I forms in the blood of patients with unstable angina pectoris

Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry

“…Patient 2, however, appeared to also have cTnT complexes present. These serum results are in accordance with data in the literature (12,(15)(16)(17)(18). The methods used to date have allowed only indirect identification of the cTnI binding partners.…”

“…In addition to complex formation with the other troponins [cTnT and cardiac troponin C (cTnC)] (12,(15)(16)(17)(18), cTnI can undergo proteolysis (8 -10, 16 ), phosphorylation (10,19,20 ), and oxidation (12 ). All of these cTnI modifications, occurring either in the myocardium before release or after release in the bloodstream, will determine the specific forms of the analyte present in a human body fluid, some of which may be directly linked to disease etiology, severity, and time after symptom onset (10, 13, 15, 20 -22 ).…”

“…Several attempts have been undertaken to identify the exact forms of cardiac troponins in the blood of patients with AMI (10,12,(15)(16)(17)(18) or unstable angina (18,22 ) or who are undergoing coronary artery bypass graft surgery (20 ). Although these studies provided indirect evidence that cTnI is predominantly present as binary cTnI-cTnC or ternary cTnI-cTnC-cTnT complex and, in addition, extensively modified, as of yet, no precise information about the actual amino acid residues involved in such modifications has been published.…”

Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

“…First, it uses real-time detection, which enables independent evaluation of each binding event and acquisition of multiple binding information, in contrast to end-point ELISA that is a single result as the sum of all binding steps. This also facilitated stepwise optimization and troubleshooting and resulted in a shorter running time than that of ELISA assays (17)(18)(19). Second, the assay is antibody dependent.…”

“…On the basis of a variety of ELISA assays (which differentiate between cTnI-cTnC and cTnI-cTnT, but not between the dimeric and trimeric complexes), investigators have proposed that the primary circulating form is the cTnI-cTnC complex, at least in a limited number of patients diagnosed with unstable angina (17 ) or MI (18 -20 ). The first direct measurement of the cTnIcTnC complex was obtained by coupling immunoaffinity capture of circulating cTnI with subsequent trypsin digestion and identification of bound proteins by mass spectrometry (MS) (13 ).…”