Department of Molecular Biology and Centre for Computational and Integrative Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA, USA.
CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
Methods Mol Biol. 2023;2690:137-147. doi: 10.1007/978-1-0716-3327-4_14.
Identification of protein-protein interactions (PPIs) and protein kinase substrates is fundamental for understanding how proteins exert biological functions with their partners and targets. However, it is still technically challenging, especially for transient and weak interactions involved in most cellular processes. The proximity-tagging systems enable capturing snapshots of both stable and transient PPIs. In this chapter, we describe in detail the methodology of a novel proximity-based labeling approach, PUP-IT (pupylation-based interaction tagging), to identify PPIs using a protoplast transient expression system. We have successfully identified potential kinase substrates by targeted screening and tandem mass spectrometry analysis.
鉴定蛋白质-蛋白质相互作用（PPIs）和蛋白激酶底物对于理解蛋白质如何与其伴侣和靶标发挥生物学功能至关重要。然而，这仍然具有技术挑战性，特别是对于涉及大多数细胞过程的瞬时和弱相互作用。邻近标记系统能够捕获稳定和瞬时 PPI 的快照。在本章中，我们详细描述了一种新型基于邻近标记的方法 PUP-IT（基于泛素化的相互作用标记）的方法学，该方法使用原生质体瞬时表达系统来鉴定 PPI。我们已经通过靶向筛选和串联质谱分析成功鉴定了潜在的激酶底物。
