Hendriks Ivo A, Vertegaal Alfred C O
Department of Molecular Cell Biology, Leiden University medical Center, Albinusdreef 2, 2300, Leiden, Netherlands.
Methods Mol Biol. 2016;1475:171-93. doi: 10.1007/978-1-4939-6358-4_13.
Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days.
基于质谱的方法正越来越频繁地被使用，以促进新型SUMO靶蛋白的鉴定，并阐明细胞应激反应中SUMO化修饰的动态变化。在此，我们描述了一种可靠的方法，使用无标记方法来鉴定SUMO靶蛋白，并对SUMO化修饰动态进行相对定量。该方法依赖于在哺乳动物细胞系或模式生物中低水平表达的十组氨酸（His10）标签SUMO。His10标签允许对SUMO化修饰蛋白进行单步、高产率和高纯度富集，然后将其消化并通过高分辨率质谱进行分析。运行匹配和集成在免费的MaxQuant软件中的无标记定量允许进行高速率和高精度的定量，为费力的样品或细胞标记技术提供了强大的替代方案。本文所述方法无需样品分级分离即可鉴定超过1000种SUMO靶蛋白，并表征其SUMO化修饰动态。从总裂解物开始的纯化过程约4天即可完成。
