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Unbiased Identification of Proteins Covalently Modified by Complex Mixtures of Peroxidized Lipids Using a Combination of Electrophoretic Mobility Band Shift with Mass Spectrometry
- Source :
- Antioxidants, Vol 7, Iss 9, p 116 (2018)
- Publication Year :
- 2018
- Publisher :
- MDPI AG, 2018.
-
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.
Details
- Language :
- English
- ISSN :
- 20763921
- Volume :
- 7
- Issue :
- 9
- Database :
- Directory of Open Access Journals
- Journal :
- Antioxidants
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.43ac9611df3d45ed9fcc8bb403f5e583
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/antiox7090116