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Oxidized lipids play a significant role in the pathogenesis of numerous oxidative stress-related human disorders, such as atherosclerosis, obesity, inflammation, and autoimmune diseases. Lipid peroxidation, induced by reactive oxygen and nitrogen species, yields a high variety of modified lipids. Among them, carbonylated lipid peroxidation products (oxoLPP), formed by oxidation of the fatty acid moiety yielding aldehydes or ketones (carbonyl groups), are electrophilic compounds that are able to modify nucleophilic substrates like proteins, nucleic acid, and aminophospholipids. Some carbonylated phosphatidylcholines possess even pro-inflammatory activities. However, little is known about oxoLPP derived from other phospholipid (PL) classes. Here, we present a new analytical strategy based on the mass spectrometry (MS) of PL-oxoLPP derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). Shotgun MS revealed many oxoLPP derived from in vitro oxidized glycerophosphatidylglycerols (PG, 31), glycerophosphatidylcholine (PC, 23), glycerophosphatidylethanolamine (PE, 34), glycerophosphatidylserines (PS, 7), glycerophosphatidic acids (PA, 17), and phosphatidylinositiolphosphates (PIP, 6) vesicles. This data were used to optimize LipidXplorer-assisted identification, and a python-based post-processing script was developed to increase both throughput and accuracy. When applied to full lipid extracts from rat primary cardiomyocytes treated with peroxynitrite donor SIN-1, ten PL-bound oxoLPP were unambiguously identified by LC-MS, including two PC-, two PE-, one PG-, two PS-, and three PA-derived species. Some of the well-known carbonylated PC were detected, while most PL-oxoLPP were shown for the first time. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

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]