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Antioxidants 2018, 7(9), 116; https://doi.org/10.3390/antiox7090116

Unbiased Identification of Proteins Covalently Modified by Complex Mixtures of Peroxidized Lipids Using a Combination of Electrophoretic Mobility Band Shift with Mass Spectrometry

Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz, Humboldtstrasse 46, 8010 Graz, Austria
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Received: 12 July 2018 / Revised: 27 August 2018 / Accepted: 29 August 2018 / Published: 30 August 2018
(This article belongs to the Special Issue Antioxidants and Second Messengers of Free Radicals)
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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. View Full-Text
Keywords: lipid oxidation; oxidized phospholipids; lipid-protein adducts; electrophoretic mobility shift assay; gel-shift; proteomics; mass spectrometry lipid oxidation; oxidized phospholipids; lipid-protein adducts; electrophoretic mobility shift assay; gel-shift; proteomics; mass spectrometry
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Gesslbauer, B.; Kuerzl, D.; Valpatic, N.; Bochkov, V.N. Unbiased Identification of Proteins Covalently Modified by Complex Mixtures of Peroxidized Lipids Using a Combination of Electrophoretic Mobility Band Shift with Mass Spectrometry. Antioxidants 2018, 7, 116.

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