Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
J Proteomics. 2017 Aug 23;166:101-114. doi: 10.1016/j.jprot.2017.07.006. Epub 2017 Jul 20.
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 1.4μM [C]-linoleic acid (LA) until the LA to [C]-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]-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]-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 and C aldehyde isomers. Using the current methodology, we identified the major lipid hydroperoxide-mediated modifications to proteins in MCF-7 cells without the need for chemical labeling or further affinity purification.
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.
由于脂质氢过氧化物介导的蛋白质修饰可能参与各种退行性疾病，因此它们受到越来越多的关注。这些生物学效应归因于脂质过氧化衍生的醛与蛋白质的亲核位点反应的能力。在这里，我们描述了一种涉及代谢标记与基于质谱的蛋白质组学分析相结合的方法，该方法能够在细胞系统中对脂质氢过氧化物介导的蛋白质修饰进行全局筛选。用 1.4μM [C]-亚油酸（LA）孵育 MCF-7 细胞，使 LA 与 [C]-LA 的比例达到 1:1，从而对 MCF-7 细胞的脂类组进行标记。这种方法称为 SILFAC（细胞培养中的脂肪酸稳定同位素标记）。对细胞磷脂的分析表明，[C]-LA 被定量掺入。用钙离子载体和 L-抗坏血酸使标记的细胞产生氧化应激，这两种物质促进细胞 LA 和 [C]-LA 中反应性醛的产生。用胰蛋白酶提取和消化蛋白质后，用同位素图案依赖 MS 分析用 C 和 C 醛异构体 1:1 比例修饰的肽。使用当前的方法，我们在无需化学标记或进一步亲和纯化的情况下，鉴定了 MCF-7 细胞中主要的脂质氢过氧化物介导的蛋白质修饰。
脂质过氧化衍生的醛（LPDAs），如 4-氧-2（E）-壬烯醛和 4-羟基-2（E）-壬烯醛，可以与蛋白质和肽迅速反应，产生多种共价修饰和交联，导致蛋白质功能障碍和基因调控改变。因此，已经开发了各种分析方法来检测和表征 LPDAs 介导的蛋白质修饰。然而，大多数方法不是针对 LPDA 修饰或针对目标醛修饰的蛋白质设计的。在这里，我们描述了细胞培养中的脂肪酸稳定同位素标记（SILFAC）与基于同位素图案的 MS 蛋白质组学策略的结合，为鉴定脂质氢过氧化物介导的蛋白质修饰提供了一种全局筛选工具。
