Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.
Nat Methods. 2013 Aug;10(8):730-6. doi: 10.1038/nmeth.2557. Epub 2013 Jul 7.
Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.
亲和纯化结合质谱(AP-MS)是一种广泛用于鉴定蛋白质-蛋白质相互作用的方法。然而,对于任何给定的感兴趣的蛋白质,确定鉴定出的多肽中哪些是真正的相互作用蛋白,而哪些是背景污染物(例如,与固相载体、亲和试剂或表位标签相互作用的蛋白质)是一项具有挑战性的任务。标准方法是使用一个或多个阴性对照纯化来鉴定非特异性相互作用,但当可用对照有限时,许多小规模的 AP-MS 研究无法捕获完整、准确的背景蛋白集。幸运的是,阴性对照在很大程度上与诱饵无关。因此,从多个 AP-MS 研究中聚合阴性对照可以增加覆盖范围,并改善与特定实验方案相关的背景特征。在这里,我们介绍了亲和纯化的污染物库(CRAPome),并描述了它在蛋白质-蛋白质相互作用评分中的应用。该存储库(目前可用于人类和酿酒酵母)和计算工具可在 http://www.crapome.org/ 免费获取。