Stable Isotope Labeling by Fatty Acids in Cell Culture (SILFAC) Coupled with Isotope Pattern Dependent Mass Spectrometry for Global Screening of Lipid Hydroperoxide-Mediated Protein Modifications

Lipid hydroperoxide-mediated modifications of proteins are receiving increasing attention because of their possible involvement in various degenerative diseases. These biological effects are attributed to the ability of lipid peroxidation-derived aldehydes to react with the nucleophilic sites of proteins. Here we describe a methodology involving metabolic labeling coupled with mass spectrometry-based proteomic analysis that enables global screening of lipid hydroperoxide-mediated protein modifications in a cell system. The lipidome of MCF-7 cells was labeled by incubating the cells with 1.4 mu M [(13)C18]-linoleic acid (IA) until the LA to [(13)C18]-LA ratio became 1:1. This approach was termed SILFAC (stable isotope labeling by fatty acids in cell culture). Analysis of the cellular phospholipids indicated that [C-13(18)]-LA was incorporated quantitatively. The labeled cells were subjected to oxidative stress using a calcium ionophore and L-ascorbic acid, which promote the generation of reactive aldehydes from cellular LA and [C-13(18)]-LA. After protein extraction and digestion with trypsin, isotope pattern dependent MS was used to analyze peptides modified by 1:1 ratios of the C-12 and C-13 aldehyde isomers. Using the current methodology, we identified the major lipid hydroperoxide-mediated modifications to proteins in MCF7 cells without the need for" chemical labeling or further affinity purification.Significance: Lipid peroxidation-derived aldehydes (LPDAs) such as 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can readily react with proteins and peptides to produce a variety of covalent modifications and cross linkages, resulting in protein dysfunction and altered gene regulation. Various analytical approaches have therefore been developed to detect and characterize protein modifications mediated by LPDAs. However, most of the methods are not specific for LPDA modifications or designed for proteins modified by a target aldehyde. Here we describe the coupling of stable isotope labeling by fatty acids in cell culture (SILFAC) with an isotope pattern dependent MS-based proteomic strategy to provide a global screening tool for the identification of lipid hydroperoxide-mediated protein modifications. (C) 2017 Elsevier B.V. All rights reserved.