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Abstract：
Complexome Profiling (CP) combines size separation, by electrophoresis or other means, of native multimeric complexes with protein identification by mass spectrometry (MS). Peptide MS analysis of the multiple fractions in which the sample is separated, results in the creation of protein abundance profiles in function of molecular size, providing a visual output of the assembly status of a group of proteins of interest. Stable isotope labeling by amino acids in cell culture (SILAC) is an established quantitative proteomics technique that allows duplexing in the MS analysis as well as the comparison of relative protein abundances between the samples, which are processed and analyzed together. Combining SILAC and CP permitted the direct comparison of migration and abundance of the proteins present in the mitochondrial respiratory chain complexes in two different samples. This analysis, however, introduced a level of complexity in data processing for which bioinformatic tools had to be developed in order to generate the normalized protein abundance profiles. The advantages and challenges of using of this type of analysis for the characterization of two cell lines carrying pathological variants in MT-CO3 and MT-CYB is reviewed. An additional unpublished example of SILAC-CP of a cell line with an in-frame 18-bp deletion in MT-CYB is presented. In these cells, in contrast to other MT-CYB deficient models, a small proportion of complex III2 is formed and it is found associated with fully assembled complex I. This analysis also revealed a profuse accumulation of assembly intermediates containing complex III subunits UQCR10 and CYC1, as well as a profound early-stage complex IV assembly defect.
摘要：
ComplexomeProfiling(CP)结合了大小分离，通过电泳或其他方式，通过质谱（MS）进行蛋白质鉴定的天然多聚体复合物。分离样品的多个级分的肽MS分析，结果在分子大小的函数中创建蛋白质丰度谱，提供一组感兴趣的蛋白质的组装状态的视觉输出。通过细胞培养中的氨基酸进行稳定同位素标记（SILAC）是一种已建立的定量蛋白质组学技术，可在MS分析中进行双工，并比较样品之间的相对蛋白质丰度。一起处理和分析。SILAC和CP的组合允许直接比较两种不同样品中线粒体呼吸链复合物中存在的蛋白质的迁移和丰度。这个分析,然而,在数据处理中引入了一定程度的复杂性，必须开发生物信息学工具才能生成归一化的蛋白质丰度谱。回顾了使用这种类型的分析来表征MT-CO3和MT-CYB中携带病理变体的两种细胞系的优势和挑战。提供了在MT-CYB中具有框内18-bp缺失的细胞系的SILAC-CP的另一个未公开的例子。在这些细胞中,与其他MT-CYB缺乏模型相比，形成了一小部分复合物III2，并发现它与完全组装的复合物I有关。该分析还揭示了包含复合物III亚基UQCR10和CYC1的组装中间体的大量积累，以及深刻的早期复合物IV组装缺陷。
