Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS).
Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS).
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DOI：
10.1074/mcp.m114.041012
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发表时间：
2015-01
期刊：
影响因子：
--
通讯作者：
Mann M
中科院分区：
文献类型：
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作者：
Keilhauer EC;Hein MY;Mann M
蛋白质之间的相互作用是理解生物过程的基础。亲和纯化-质谱联用(AP-MS)是最有前景的研究方法之一。以前，尽可能多地提纯复合体，然后经常鉴定单独的凝胶条带。然而，今天的质谱仪是高度灵敏的，强大的定量蛋白质组学策略可以区分真正的相互作用体和背景结合物。在这里，我们描述了一种用于研究蛋白质-蛋白质相互作用的高效亲和富集质谱方法，在该方法中，没有尝试将络合物纯化到均一性。相反，我们开发了分析方法，这些方法利用了在大量非特定背景结合物的背景下对交互作用进行特定浓缩的优势。我们在萌芽酵母中对内源表达的GFP标记蛋白及其相互作用因子进行单步亲和浓缩，然后进行单次运行、基于强度的无标记定量LC-MS/MS分析。每个下拉菜单包含大约2000个背景活页夹，从令人不安的污染物到新的数据分析策略中的关键元素进行了重新解释。首先，背景为准确的归一化服务。其次，相互作用的蛋白质不是通过与单个未标记的对照菌株进行比较来鉴定的，而是通过与其他标记的菌株进行比较来鉴定的。第三，潜在的相互作用通过它们在所有样本上的强度分布进一步得到验证。我们展示了我们的声发射-质谱仪方法的力量，使用几个众所周知的和具有挑战性的不同丰度的酵母复合体。AE-MS不仅高效、坚固，而且性价比高，适用范围广，可以在任何可以使用高分辨率质谱仪的实验室进行。
Protein–protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein–protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers.