A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells
A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells
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DOI：
10.1074/mcp.m110.004796
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发表时间：
2011-02-01
影响因子：
7
通讯作者：
Thibault, Pierre
中科院分区：
文献类型：
--
作者：
Galisson, Frederic;Mahrouche, Louiza;Thibault, Pierre
小泛素相关修饰物(SUMO)是一小群蛋白质，它们可逆地附着在蛋白质底物上，以改变其功能。哺乳动物细胞中蛋白质SUMO化及其修饰位点的大规模鉴定是一个巨大的挑战，因为体内底物的数量相对较少，而且这种修饰的动态性质。我们在这里报道了一种新的蛋白质组学方法来选择性地丰富和鉴定人类细胞中的相扑偶联物。我们在HEK293细胞中稳定地表达了不同的SUMO类似物，每个都含有一个His 6标签和一个位于C末端的具有战略位置的胰酶裂解位点，以便于亲和富集法和质谱法对SUMO化多肽的回收和鉴定。具有短相扑残基的胰蛋白酶多肽在大规模的Sumoylome实验中具有显著的优势，包括在CID和ETD激活后产生Paralog特异的片段离子，以及使用传统的数据库搜索引擎(如Mascot)识别修饰的多肽。我们确定了205个独特的蛋白质底物，以及12个相扑蛋白结合物中存在的17个精确的SUMO化位点，其中包括早幼粒白血病蛋白上的三个新位点(Lys-380、Lys-400和Lys-497)。对未经处理和三氧化二砷处理的细胞的纯化核提取液进行的无标记定量蛋白质组学分析表明，所有已鉴定的早幼粒细胞白血病的SUMO化位点在刺激下都发生了不同的SUMO化。分子与细胞蛋白质组学10：10.1074/mcp.M110.004796，1-15,2011年。
The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His 6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.004796, 1-15, 2011.