Banks Charles A S, Kong Stephanie E, Washburn Michael P
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Protein Expr Purif. 2012 Dec;86(2):105-19. doi: 10.1016/j.pep.2012.09.007. Epub 2012 Sep 24.
Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.
对蛋白质复合物进行表征并鉴定其亚基，有助于我们了解许多体内过程所涉及的机制。蛋白质组学研究可以识别蛋白质的结合伴侣，这有助于深入了解蛋白质复合物的功能及其调控方式。此外，还可以研究生物体内蛋白质复合物的组成随时间、位置的变化，或生物体对环境变化的反应。分离复合物并鉴定其成分的方法有很多。本综述将重点介绍使用亲和纯化分离复合物的方法，并讨论生物化学家在采用多维蛋白质鉴定技术（MudPIT）进行质谱分析分离蛋白质复合物时应牢记的问题(1)。通过质谱分析蛋白质复合物通常需要纯化蛋白质复合物的生物化学家或生物学家与分析样品的蛋白质组学专家共同协作——为了实现富有成效的合作，这些专业团队熟悉彼此学科的基本原理会很有帮助。有鉴于此，我们首先综述可用于制备待分析复合物的各种亲和纯化方法，然后简要介绍MudPIT质谱分析和数据分析的原理。在此基础上，我们将讨论如何整合和优化这些技术，并提出在制备用于蛋白质鉴定的纯化样品、进行质谱分析以及分析所得数据时需要考虑的要点。
