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Recent research indicates that oxidative stress (OS) affects all levels of cellular organization and induce perturbation in gene expression, epigenetic regulation, mRNA and protein levels, and cellular metabolism. Oxidative modifications of proteins and lipids represent another level of complexity in cellular regulation supported by numerous studies demonstrating how these modifications regulate gene expression and cellular signaling pathways. However, it is almost impossible to specify the input and (patho)physiological consequences of OS using data from a single modification level. Nowadays, it is clear that only a holistic approach, as suggested by systems biology, will provide an integrated view on OS-related cellular regulation and pathologies by considering all known OS determinants, identifying new ones, and establishing functional links among them. As a first effort to study OS via redox systems biology, we applied a multi-omics approach to characterize the impact of OS on cardiomyocytes (CM) using a dynamic model of nitrosative stress. Reactive carbonylated lipids, hydroxylated and truncated phospholipids, nitrated fatty acids, and oxysterols were quantified relative to a control in CM lipid extracts after 15, 30, 70 min, and 16 h of OS. Simultaneously, the protein fraction was analyzed for a wide range of post-translational modifications, such as phosphorylation, carbonylation, cysteine oxidation, and nitrosylation. Omics data were combined and supported by biochemical and microscopy studies on oxidation dynamics, spatial distribution, and functional effects. Thus, the combination of lipidomics, data-driven proteomics, and systems biology integration allowed the identification of more than 200 proteins modified by reactive lipid peroxidation products of which many were involved in calcium signaling pathways, regulation of actin cytoskeleton, focal adhesion, and phosphatidylinositol signaling system. Biochemical and microscopy studies further confirmed OS-derived impairment of Ca-signaling and cytoskeletal protein distribution. [ABSTRACT FROM AUTHOR]

Within the wide range of oxidative modifications, "carbonylation" formed by the incorporation of aldehyde/keto groups, is commonly studied due to its role in cell physiology and as prospective biomarkers for numerous disorders. Despite close biochemical and physiological links between protein and lipid carbonylation, these two types of modifications are rarely addressed simultaneously in a single study. In nitrosative stress cell model we investigated levels of protein and lipid carbonylation and addressed the main modified species by combining LC-MS, biochemical, and microscopy studies. The influence of nitrosative stress on carbonylation of proteins and lipids was investigated for primary cardiomyocytes treated with SIN-1 for different time intervals. Lipid carbonylation was quantified by RPC-ESI-MS/MS. The results demonstrate dynamic generation, degradation and adduct formations of 25 different species including alkanals, alkenals, alkadienals, alkatrienals and oxo-carboxylic acids. Several new PL-bound aldehydes were present exclusively after a long incubation period. Carbonylated proteins were identified after aldehyde reactive probe derivatization, affinity enrichment and RPC-ESI-MS/MS. More than 200 proteins were identified and evaluated by systems biology to deduce the biological significance of the protein modifications. The protein carbonylation degree was verified using oxyblot and correlated with changes in 20 S/26 S proteasome activities. Furthermore, a new fluorescence microscopy based technique to stain carbonylated biomolecules was developed and compared with conventional DNPH-based immunocytochemistry. Subcellular localization of carbonylated species was investigated using mitochondrial and ER-specific co-localization experiments. Thus, the combination of lipidomics, proteomics, biochemical techniques, and microscopy imaging revealed a complex molecular pattern of "carbonylation stress" in the studied nitrosative stress cell model. [ABSTRACT FROM AUTHOR]