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Reactive oxygen and nitrogen species (ROS/RNS) play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15 min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), and decreased to values close to control after 16 h. Total (lipids+proteins) vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion channels was further confirmed., Graphical abstract fx1, Highlights • Different dynamics for lipid and protein carbonylation upon nitroxidative stress. • Shift of carbonyls from lipids to proteins within first 30 min. • Neutral and oxidized lipids increased in the presence of lysosomal inhibitors. • Electrophilic lipids identified and relatively quantified by LC-MS/MS. • Cytoskeleton, ECM and ion channel proteins are the main modification targets.