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

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-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 activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to allow the use of the protein-of-interest 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 protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control to match expression levels for proximity labeling proteomics.蛋白质-蛋白质相互作用在细胞稳态与功能的各个层面均发挥关键生物学作用。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难题，现已迅速成为该领域的当前前沿技术。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自切割肽与非切割突变体，实现实验与对照TurboID体系中目的蛋白的精准应用。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，以制备可用于瞬时表达与稳定整合的质粒。为展示我们的T2A Split-link设计，我们通过TurboID邻近标记技术，将其应用于鉴定糖皮质激素受体、严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核衣壳蛋白与NSP7蛋白的相互作用。研究结果表明，我们的T2A split/link设计可提供适配的对照体系，以匹配邻近标记蛋白质组学实验中的表达水平。