Ori Alessandro, Andrés-Pons Amparo, Beck Martin
European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstr. 1, 69117, Heidelberg, Germany.
Methods Cell Biol. 2014;122:117-46. doi: 10.1016/B978-0-12-417160-2.00006-0.
Accurate knowledge of the stoichiometry of protein complexes is a crucial prerequisite for understanding their structure and function. To purify or enrich large and intricate protein complexes such that their structure is preserved and to absolutely quantify all of their protein components is an enormous technical challenge. In this chapter, we describe how to purify nuclear envelopes from human tissue culture cells that are highly enriched for nuclear pore complexes. We use the nuclear pore as an example to discuss how the structural preservation of such preparations can be controlled. Furthermore, we give a practical guide how to develop and employ targeted proteomic assays for both, the absolute quantification of stoichiometries and the relative quantification of protein complex composition across multiple biological conditions. The concept discussed here is universally applicable to any protein complex.
准确了解蛋白质复合物的化学计量是理解其结构和功能的关键前提。纯化或富集大型复杂蛋白质复合物，使其结构得以保留，并对其所有蛋白质成分进行绝对定量，是一项巨大的技术挑战。在本章中，我们描述了如何从高度富集核孔复合物的人组织培养细胞中纯化核膜。我们以核孔为例，讨论如何控制此类制剂的结构保存。此外，我们提供了一份实用指南，介绍如何开发和应用靶向蛋白质组学分析方法，用于绝对定量化学计量以及在多种生物学条件下对蛋白质复合物组成进行相对定量。这里讨论的概念普遍适用于任何蛋白质复合物。
