Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Mol Cell Proteomics. 2010 Aug;9(8):1729-41. doi: 10.1074/mcp.M110.001446. Epub 2010 Jun 8.
Many fundamental processes in the cell are performed by complex macromolecular assemblies that comprise a large number of proteins. Numerous macromolecular assemblies are structurally rather fragile and may suffer during purification, resulting in the partial dissociation of the complexes. These limitations can be overcome by chemical fixation of the assemblies, and recently introduced protocols such as gradient fixation during ultracentrifugation (GraFix) offer advantages for the analysis of fragile macromolecular assemblies. The irreversible fixation, however, is thought to render macromolecular samples useless for studying their protein composition. We therefore developed a novel approach that possesses the advantages of fixation for structure determination by single particle electron microscopy while still allowing a correlative compositional analysis by mass spectrometry. In this method, which we call "electron microscopy carbon film-assisted digestion", macromolecular assemblies are chemically fixed and then adsorbed onto electron microscopical carbon films. Parallel, identically prepared specimens are then subjected to structural investigation by electron microscopy and proteomics analysis by mass spectrometry of the digested sample. As identical sample preparation protocols are used for electron microscopy and mass spectrometry, the results of both methods can directly be correlated. In addition, we demonstrate improved sensitivity and reproducibility of electron microscopy carbon film-assisted digestion as compared with standard protocols. We show that sample amounts of as low as 50 fmol are sufficient to obtain a comprehensive protein composition of two model complexes. We suggest our approach to be an optimization technique for the compositional analysis of macromolecules by mass spectrometry in general.
许多细胞中的基本过程都是由包含大量蛋白质的复杂大分子组装体来执行的。许多大分子组装体在结构上相当脆弱，在纯化过程中可能会受到损伤，导致复合物的部分解离。这些限制可以通过化学固定组装体来克服，最近引入的一些方法，如超速离心过程中的梯度固定（GraFix），为分析脆弱的大分子组装体提供了优势。然而，这种不可逆的固定被认为会使大分子样品无法用于研究其蛋白质组成。因此，我们开发了一种新的方法，该方法结合了固定对于单颗粒电子显微镜结构测定的优势，同时仍然允许通过质谱进行相关的组成分析。在这种被我们称为“电子显微镜碳膜辅助消化”的方法中，大分子组装体先被化学固定，然后吸附到电子显微镜的碳膜上。然后，平行制备的相同样本分别进行电子显微镜结构研究和消化样品的质谱分析的蛋白质组学分析。由于电子显微镜和质谱分析使用相同的样品制备方案，因此两种方法的结果可以直接相关。此外，我们证明了电子显微镜碳膜辅助消化与标准方案相比，具有更高的灵敏度和重现性。我们表明，低至 50fmol 的样品量足以获得两个模型复合物的综合蛋白质组成。我们建议我们的方法是一般通过质谱进行大分子组成分析的优化技术。
