Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

PPIs play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spec-based proteomics overcomes challenges typically associated with other methods, and has quickly become the current state-of-the-art in the field. Nevertheless, tight control of proximity labeling enzymatic acitivty a,d expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleavage mutant to allow the use of the POI in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we build a GOlden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A split-link design, we applied it to identify PPIs of GR, NCAP, and NSP7 by TurboID proximity labeling proteomics. Our results demisntrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field.

蛋白质-蛋白质相互作用（Protein-Protein Interactions, PPIs）在细胞稳态与功能的各个层面均发挥重要生物学作用。基于邻近标记质谱的蛋白质组学克服了其他方法通常面临的技术挑战，现已迅速成为该领域当前最先进的技术方案。然而，严格调控邻近标记酶的活性与表达水平，对于精准鉴定蛋白质相互作用因子至关重要。本研究利用T2A自剪切肽与非剪切突变体，使目标蛋白（Protein of Interest, POI）可同时应用于实验组与对照组的TurboID实验体系。为实现便捷高效的质粒组装流程，我们构建了Golden Gate模块化克隆系统，以制备用于瞬时表达与稳定整合的质粒载体。为验证我们的T2A分裂连接体设计，我们将其应用于通过TurboID邻近标记蛋白质组学鉴定GR、NCAP与NSP7的蛋白质相互作用。研究结果表明，我们开发的T2A分裂连接体可提供契合该领域已有质控要求的理想对照方案。