In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.
In vivo identification of human small ubiquitin-like modifier polymerization sites by high accuracy mass spectrometry and an in vitro to in vivo strategy.
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DOI：
10.1074/mcp.m700173-mcp200
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发表时间：
2008-01
期刊：
影响因子：
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通讯作者：
Vertegaal ACO
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文献类型：
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作者：
Matic I;van Hagen M;Schimmel J;Macek B;Ogg SC;Tatham MH;Hay RT;Lamond AI;Mann M;Vertegaal ACO
多聚泛素链的长度和精确连接对其生物活性是重要的。虽然其他泛素样蛋白有可能形成聚合链，但它们的体内鉴定具有挑战性，并且它们的功能作用尚不清楚。脊椎动物表达三种小的泛素样修饰物，SUMO-1，SUMO-2和SUMO-3。成熟的SUMO-2和SUMO-3几乎相同，并且含有SUMO-1中缺失的SUMO化的内部共有位点。结合最先进的质谱与翻译后修饰的“体外到体内”策略，我们提供了直接的证据，即SUMO-1，SUMO-2和SUMO-3通过SUMO-2和SUMO-3中SUMO化的内部共有位点在细胞中形成混合链。在体外，SUMO聚合物的链长可以通过改变SUMO-1和SUMO-2的相对量来影响。所开发的方法是通用的，可以适应于复杂的样品中的其他sumoylation网站的识别。
The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an “in vitro to in vivo” strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.