Division of Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands; Netherlands Proteomics Center, Utrecht, The Netherlands.
Division of Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands; Netherlands Proteomics Center, Utrecht, The Netherlands.
Mol Cell Proteomics. 2023 Nov;22(11):100657. doi: 10.1016/j.mcpro.2023.100657. Epub 2023 Oct 6.
Mitochondria are densely packed with proteins, of which most are involved physically or more transiently in protein-protein interactions (PPIs). Mitochondria host among others all enzymes of the Krebs cycle and the oxidative phosphorylation pathway and are foremost associated with cellular bioenergetics. However, mitochondria are also important contributors to apoptotic cell death and contain their own genome indicating that they play additionally an eminent role in processes beyond bioenergetics. Despite intense efforts in identifying and characterizing mitochondrial protein complexes by structural biology and proteomics techniques, many PPIs have remained elusive. Several of these (membrane embedded) PPIs are less stable in vitro hampering their characterization by most contemporary methods in structural biology. Particularly in these cases, cross-linking mass spectrometry (XL-MS) has proven valuable for the in-depth characterization of mitochondrial protein complexes in situ. Here, we highlight experimental strategies for the analysis of proteome-wide PPIs in mitochondria using XL-MS. We showcase the ability of in situ XL-MS as a tool to map suborganelle interactions and topologies and aid in refining structural models of protein complexes. We describe some of the most recent technological advances in XL-MS that may benefit the in situ characterization of PPIs even further, especially when combined with electron microscopy and structural modeling.
线粒体中富含蛋白质，其中大多数蛋白质直接或间接地参与蛋白质-蛋白质相互作用（PPIs）。线粒体是三羧酸循环和氧化磷酸化途径所有酶的宿主，与细胞生物能量学关系最为密切。然而，线粒体也是细胞凋亡的重要贡献者，并且含有自己的基因组，这表明它们在生物能量学之外的过程中也发挥着重要作用。尽管通过结构生物学和蛋白质组学技术在鉴定和描述线粒体蛋白复合物方面做出了巨大努力，但仍有许多 PPIs 难以捉摸。其中一些（膜嵌入）PPIs 在体外不太稳定，阻碍了大多数当代结构生物学方法对其进行特征描述。特别是在这些情况下，交联质谱（XL-MS）已被证明对原位深入表征线粒体蛋白复合物非常有价值。在这里，我们强调了使用 XL-MS 分析线粒体中全蛋白质组 PPIs 的实验策略。我们展示了原位 XL-MS 作为一种工具的能力，用于绘制亚细胞器相互作用和拓扑结构，并有助于改进蛋白复合物的结构模型。我们描述了 XL-MS 中一些最新的技术进步，这些进步可能会进一步受益于 PPIs 的原位特征描述，特别是与电子显微镜和结构建模相结合时。
