Affinity purification coupled with mass spectrometry (AP–MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP−MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP–MS and BioID data has been shown to comprehensively characterize a protein’s molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP–MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform (http://proteomics.fi) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP–MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.

亲和纯化与质谱联用（AP-MS）和邻近依赖性生物素化鉴定（BioID）方法为相互作用蛋白质组学研究做出了重要贡献。AP-MS可以鉴定稳定复合物中的蛋白质，而BioID可以标记并鉴定与诱饵非常接近的蛋白质，从而导致重叠但又不同的蛋白质鉴定。已显示AP-MS和BioID数据的整合可以全面表征蛋白质的分子环境，但是在细胞系生成和蛋白质纯化方面，同时使用两种方法进行相互作用组分析仍然是劳动和资源密集型工作。因此，我们开发了多种方法结合（MAC）标签工作流程，它允许使用单个构建体以及几乎相同的蛋白质纯化和质谱（MS）鉴定程序进行AP-MS和BioID分析。我们已将MAC标签工作流程应用于亚细胞标记物的选择，以提供细胞蛋白质相互作用组图景的全局视图。可通过我们的在线平台（http://proteomics.fi）访问此定位数据库，以根据目标蛋白质（POI）识别的相互作用因子预测其在细胞中的定位。在该协议中，我们为MAC标签工作流程提供了详细的三阶段过程：（1）为MAC标签POI生成细胞系；（2）并行进行AP–MS和BioID蛋白质纯化，然后进行MS分析；（3）使用我们的本地化可视化平台进行蛋白质相互作用数据分析，数据过滤和可视化。