Highly accurate protein complex retrieval by affinity enrichment MS rather than affinity purification MS

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, today 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 (AE-MS) 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. Firstly, the background serves for accurate normalization. Secondly, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Thirdly, 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 feasible to perform in any laboratory.

蛋白质-蛋白质相互作用是解析生物学过程的核心基石。亲和纯化联用质谱（Affinity Purification coupled to Mass Spectrometry，AP-MS）是研究此类相互作用最具前景的方法之一。此前，研究人员往往尽可能纯化复合物，随后常通过对凝胶电泳单一条带进行鉴定来获取相关信息。然而如今，质谱仪灵敏度大幅提升，且已涌现出强大的定量蛋白质组学策略，可将真实相互作用蛋白与背景结合蛋白区分开来。
本文介绍一种高性能亲和富集-质谱联用（Affinity Enrichment-Mass Spectrometry，AE-MS）方法，用于研究蛋白质-蛋白质相互作用，该方法无需将复合物纯化至均质状态。与之不同的是，我们开发了分析方法，可在大量非特异性背景结合蛋白存在的前提下，利用相互作用蛋白的特异性富集特性完成分析。我们在出芽酵母中对绿色荧光蛋白（GFP）标记的内源表达蛋白及其相互作用蛋白开展单步亲和富集，随后通过单次上机、基于信号强度的无标记定量液相色谱-串联质谱（LC-MS/MS）分析。每个亲和下拉实验样本中约包含2000种背景结合蛋白，在新型数据分析策略中，这些曾被视为干扰性污染物的蛋白已被重新定义为关键分析要素。其一，背景结合蛋白可用于实现精准归一化；其二，相互作用蛋白的鉴定并非仅与单一未标记对照菌株进行比对，而是与其他标记菌株展开对比；其三，潜在相互作用蛋白可通过其在所有样本中的信号强度分布特征得到进一步验证。
我们选用多种丰度各异的经典且具有挑战性的酵母复合物，验证了AE-MS方法的优异性能。该方法不仅高效稳定、成本低廉，还具备广泛的适用性，且可在任意实验室中便捷开展。