Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, China.
PLoS One. 2013 Oct 2;8(10):e76399. doi: 10.1371/journal.pone.0076399. eCollection 2013.
O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.
O-连接的β-N-乙酰氨基葡萄糖（O-GlcNAc）是一种重要的翻译后修饰（PTM），由单个 N-乙酰氨基葡萄糖通过 O-β-糖苷键连接到丝氨酸和苏氨酸残基上组成。O-GlcNAc 糖基化发生在来自几乎所有功能类别的核和细胞质蛋白上。然而，对于在线粒体中特殊的 O-GlcNAc 化蛋白，关注甚少。在这项研究中，我们结合质谱和免疫方法，对大鼠肝线粒体中特异的 O-GlcNAc 化蛋白进行了全面探索。首先，通过免疫印迹分析清楚地表明高度纯化的线粒体蛋白发生了 O-GlcNAc 化。然后，进行β消除 followed by Michael Addition with Dithiothreitol（BEMAD）处理和 LC-MS/MS，以富集和鉴定 O-GlcNAc 化的线粒体蛋白，从而明确分配了 14 个 O-GlcNAc 化位点，映射到 11 个 O-GlcNAc 化蛋白。此外，通过电子转移解离串联质谱（ETD/MS/MS）和 Western blot 对鉴定出的 O-GlcNAc 化线粒体蛋白进行了全面验证。因此，我们的研究首次不仅确定而且验证了大鼠肝线粒体中的一些蛋白质发生了 O-GlcNAc 化。有趣的是，所有这些 O-GlcNAc 化的线粒体蛋白都是酶，其中大多数参与各种生物学过程，如尿素循环、三羧酸循环和脂质代谢，表明蛋白质 O-GlcNAc 化在线粒体功能中发挥作用。
