Updated project metadata. Myocardial infarction (MI) is the most common cause of death worldwide. Timely diagnosis and early restoration of the blood flow prevents further tissue damage. However, this sudden reperfusion can also lead to so-called ischemia/reperfusion (I/R) injury. Although the different pathological processes activated in I/R injury, like cell death and inflammation have been previously addressed, the exact pathophysiological mechanisms remain unclear. For the development of strategies that minimize myocardial damage a better understanding of the underlying alterations in protein and signaling pathways is required. In this study we aim to identify the protein signature of cardiac I/R injury, therefore we use a spatialOMx approach, combining protein mass spectrometry imaging (MSI) and a classical label-free proteomics workflow on the same tissue section, to further address the observed alterations