A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis
A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis
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DOI：
10.1038/nprot.2010.109
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发表时间：
2010-08-01
期刊：
影响因子：
14.8
通讯作者：
Burstein, Ezra
中科院分区：
文献类型：
--
作者：
Maine, Gabriel N.;Li, Haiying;Burstein, Ezra
泛素化可以对细胞蛋白质的稳定性和功能产生深远的影响。质谱 (MS) 可用于绘制目标蛋白中与泛素缀合的特定氨基酸残基。然而，蛋白质组分析所需的纯化可能具有挑战性。在本文中，我们描述了一种用于分离特定泛素化蛋白的双分子亲和纯化方案，其中亲和部分与泛素和感兴趣的靶蛋白融合。在体内泛素缀合后，蛋白质靶标获得两个亲和标签，从而可以特异性纯化其泛素修饰形式。为了防止裂解后去泛素化或相互作用的辅因子共纯化，该过程是在使用多组氨酸和生物素化标签进行蛋白质变性后进行的。使用此程序，可以有效地纯化大量的给定蛋白质的泛素化形式，其纯度足以用于 MS 分析和泛素受体位点图谱。
Ubiquitination can have profound effects on the stability and function of cellular proteins. Mass spectrometry (MS) can be used to map the specific amino acid residues that are conjugated to ubiquitin in a target protein. However, the purification required for proteomic analysis can be challenging. In this paper, we describe a bimolecular affinity purification scheme for the isolation of a specific ubiquitinated protein in which affinity moieties are fused to ubiquitin and to a target protein of interest. After ubiquitin conjugation in vivo, the protein target acquires two affinity tags, allowing the specific purification of its ubiquitin-modified forms. To prevent deubiquitination after lysis or the copurification of interacting cofactors, this procedure is performed after protein denaturation using polyhistidine and biotinylation tags. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified in large amounts of sufficient purity for MS analysis and for mapping of ubiquitin acceptor sites.