Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available providing diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis and their persistence limit their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer and coronary effluent collected after ischemia or during matched normoxic perfusion. Effluents were analysed using proteomic approaches based on 1D or 2D initial separation. Of the 459 proteins identified after ischemia with 1D separation, 320 were not detected in the control coronary effluent. Amongst these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin binding protein C (cMyBP-C) in its full-length form and as a 40 kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction and could be detected in the plasma after myocardial infarction in-vivo a profile compatible with a biomarker of AMI. 2D Fluorescence Difference In-Gel Electrophoresis (DIGE) of ischemic and control coronary effluent identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischaemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin binding protein C.