Department of Laboratory Medicine, Laboratory of Genetic Endocrine and Metabolic Disorders, Radboud University Medical Centre, Nijmegen, The Netherlands.
PLoS One. 2013 Jul 23;8(7):e68340. doi: 10.1371/journal.pone.0068340. Print 2013.
Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification-mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.
复合物图谱分析是一种新颖的技术，它使用鸟枪法蛋白质组学从分级蓝色天然电泳凝胶中建立蛋白质迁移图谱。在这里，我们提供了通过复合物图谱分析获得的 953 种蛋白质的蓝色天然电泳迁移图谱数据集。通过对线粒体核糖体复合物的分析，我们证明了它有潜力验证通过亲和纯化-质谱研究鉴定的假定蛋白质-蛋白质相互作用。将蛋白质复合物以天然状态从 HEK293 线粒体部分中提取出来，并通过蓝色天然凝胶电泳进行分离。凝胶条带被切成均匀大小的凝胶片，并通过鸟枪法蛋白质组学进行分析。随后，通过层次聚类分析对获得的蛋白质迁移图谱进行共迁移分析。该数据集有望成为从头鉴定蛋白质-蛋白质相互作用的综合资源，或者为其他研究中的候选蛋白质相互作用提供依据和优先级。为了展示我们数据集的潜在用途，我们专注于线粒体翻译机制。我们的结果表明，线粒体核糖体复合物可以通过蓝色天然凝胶电泳进行分析，至少有四个不同的复合物。对这些复合物的分析证实，先前有报道与线粒体核糖体共纯化的 24 种蛋白质确实与线粒体核糖体的亚基共迁移。几种参与线粒体内膜复合物生物发生的蛋白质与线粒体核糖体一起共迁移，这表明在人类细胞中存在共翻译组装的可能性。我们的数据还突出了一个潜在的核糖核苷酸复合物，该复合物可能含有 MRPL10、MRPL12 和 MRPL53 以及 LRPPRC 和 SLIRP。
