Abstract
Covalent modification of functionally important cell proteins by lipid oxidation products (LOPs) is a known mechanism initiating pathological consequences of oxidative stress. Identification of new proteins covalently modified by electrophilic lipids can be performed by a combination of chemical, immunological, and mass spectrometry-based methods, but requires prior knowledge either on the exact molecular structure of LOPs (e.g., 4-hydroxynonenal) or candidate protein targets. However, under the conditions of oxidative stress in vivo, a complex mixture of proteins (e.g., cytosolic proteome) reacts with a complex mixture of LOPs. Here we describe a method for detection of lipid-modified proteins that does not require an a priori knowledge on the chemical structure of LOPs or identity of target proteins. The method is based on the change of electrophoretic mobility of lipid-modified proteins, which is induced by conformational changes and cross-linking with other proteins. Abnormally migrating proteins are detected by mass spectrometry-based protein peptide sequencing. We applied this method to study effects of oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) on endothelial cells. Several known, but also many new, OxPAPC-binding proteins were identified. We expect that this technically relatively simple method can be widely applied for label-free analysis of lipid-protein interactions in complex protein samples treated with different LOPs.
Highlights
Lipid oxidation is a characteristic feature of chronic inflammation associated with a variety of pathologies including, e.g., cardiovascular and neurodegenerative disease [1,2,3,4,5]
In order to reduce the complexity of the proteome and to separate water-soluble, mainly cytosolic proteins, from membrane-associated proteins, we split the Human umbilical vein endothelial cells (HUVEC) proteome into a phosphate buffered saline (PBS)-soluble part and a PBS-insoluble/RIPA-soluble part
Both samples were subjected to Electrophoretic Mobility Band Shift Analysis (EMSA) after treatment with varying concentrations of Oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC)
Summary
Lipid oxidation is a characteristic feature of chronic inflammation associated with a variety of pathologies including, e.g., cardiovascular and neurodegenerative disease [1,2,3,4,5]. Oxidation of free or esterified polyunsaturated fatty acids (PUFAs) can be induced by reactive oxygen and nitrogen species or by multiple enzymes, generating a variety of bioactive compounds. PUFA residues in phospholipids (PLs) generates a variety of full-length and truncated reactive oxidized. Lipid oxidation products (LOPs) containing electrophilic terminal aldehyde or α,β-unsaturated carbonyl groups can Antioxidants 2018, 7, 116; doi:10.3390/antiox7090116 www.mdpi.com/journal/antioxidants. Adduction of LOPs to proteins can alter protein structure and function, and influence cellular signal transduction pathways [1,7,8]
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