National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
School of Biotechnology, Dublin City University, Dublin, Ireland.
Methods Mol Biol. 2025;2853:191-203. doi: 10.1007/978-1-0716-4104-0_13.
Ubiquitination is one of the most important post-translational modifications (PTMs) and involves the covalent attachment of ubiquitin to a lysine residue on a target protein. Despite ubiquitination playing a crucial role in regulating cellular processes, the ubiquitinated proteome has not been studied extensively in recombinant Chinese hamster ovary (CHO) cells. Moreover, ubiquitination modification in CHO cells is likely to have an impact on protein function related to the efficient productivity of biopharmaceuticals. In this chapter, we describe a comprehensive protocol for ubiquitin di-Glycine (diGly) peptide enrichment using an immunoprecipitation method from recombinant CHO cell proteins followed by Liquid chromatography-Mass spectrometry (LC-MS) analysis of the ubiquitinated proteome. The methods described are also applicable to differential ubiquitinated proteomic studies.
泛素化是最重要的翻译后修饰（PTMs）之一，涉及到泛素与靶蛋白赖氨酸残基的共价连接。尽管泛素化在调节细胞过程中起着至关重要的作用，但重组中国仓鼠卵巢（CHO）细胞中的泛素化蛋白质组尚未得到广泛研究。此外，CHO 细胞中的泛素化修饰可能会影响与生物制药高效生产相关的蛋白质功能。在本章中，我们描述了一种使用免疫沉淀法从重组 CHO 细胞蛋白中富集泛素二甘氨酸（diGly）肽的综合方案，然后进行液相色谱-质谱（LC-MS）分析泛素化蛋白质组。所描述的方法也适用于差异泛素化蛋白质组学研究。
