Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P, Sleeman J, Lamond A, Mann M
Protein & Peptide Group, European Molecular Biology Laboratory, Heidelberg, Germany.
Nat Genet. 1998 Sep;20(1):46-50. doi: 10.1038/1700.
Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors--the spliceosome--is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.
许多重要的细胞机制是由多蛋白复合物执行和调控的，例如转录和RNA加工机制、受体复合物以及细胞骨架结构。由于传统蛋白质分析方法存在困难，这些复合物中的大多数仍仅得到部分表征。DNA序列数据库的迅速扩展现在提供了模式生物的全部或部分基因序列，并且最近通过质谱进行蛋白质微表征的进展使得将这些DNA序列与功能复合物中的蛋白质联系起来成为可能。这种方法已在基因组已测序的生物体中得到证明，如芽殖酵母。在此，我们报告了使用这些方法对整个哺乳动物多蛋白复合物进行的首次表征。从mRNA前体中去除内含子的机制——剪接体——是一种大型多蛋白复合物。从剪接体的二维凝胶分离中切下的组分中，约有一半在蛋白质序列数据库中被找到。使用纳升电喷雾质谱法，其余组分通过公共表达序列标签（EST）数据库进行了鉴定和克隆。因此，现有的EST数据库已经足够完整，能够通过质谱法快速表征大型哺乳动物蛋白质复合物。
