Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo.
Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo.
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DOI：
10.1021/acs.analchem.6b03675
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发表时间：
2017-04-18
影响因子：
7.4
通讯作者：
Strieter ER
中科院分区：
文献类型：
--
作者：
Crowe SO;Rana ASJB;Deol KK;Ge Y;Strieter ER
泛素（Ub）具有广泛的功能范围，其归因于形成聚合的泛素链阵列。然而，由于其复杂的链拓扑结构，理解泛素链的确切作用是困难的。分支的泛素链是特别具有挑战性的，因为单个泛素上的多个修饰排除了标准的自下而上的蛋白质组学方法的使用。考虑到有证据表明支链调节蛋白质的稳定性，开发克服这些挑战的方法至关重要。在这项研究中，我们采用泛素链富集中下质谱法（UbiChEM-MS）来识别使用自下而上的蛋白质组学方法无法检测到的支链。具体地，我们采用来自脱泛素酶TRABID的串联泛素结合实体（TUBE）和K29选择性Npl 4锌指1（NZF 1）结构域来富集来自人细胞的链。最低限度的胰蛋白酶水解，然后高分辨率质谱分析表明，Ub链分支确实可以检测到使用两个Ub结合结构域（UBD）在内源性水平测试。我们发现，用TUBE分离的链中有10.1%含有Ub分支点，在蛋白酶体抑制后，该值上升到10.4%。电子转移解离（ETD）分析表明这些支链中存在K48。NZF 1结构域的使用表明，约4%的分离链含有分支点，对蛋白酶体抑制没有明显的依赖性。我们的研究结果证明了一种有效的策略，用于检测和表征不同细胞条件下的分支共轭物的动力学。
Ubiquitin (Ub) has a broad functional range that has been ascribed to the formation of an array of polymeric ubiquitin chains. Understanding the precise roles of ubiquitin chains, however, is difficult due to their complex chain topologies. Branched ubiquitin chains are particularly challenging, as multiple modifications on a single ubiquitin preclude the use of standard bottom-up proteomic approaches. Developing methods to overcome these challenges is crucial considering evidence suggesting branched chains regulate the stability of proteins. In this study, we employ Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS) to identify branched chains that cannot be detected using bottom-up proteomic methods. Specifically, we employ tandem ubiquitin binding entities (TUBEs) and the K29-selective Npl4 Zinc Finger 1 (NZF1) domain from the deubiquitinase TRABID to enrich for chains from human cells. Minimal trypsinolysis followed by high resolution mass spectrometric analysis reveals that Ub chain branching can indeed be detected using both Ub binding domains (UBDs) tested at endogenous levels. We find that ∼1% of chains isolated with TUBEs contain Ub branch points, with this value rising to ∼4% after proteasome inhibition. Electron-transfer dissociation (ETD) analysis indicates the presence of K48 in these branched chains. The use of the NZF1 domain reveals that ∼4% of the isolated chains contain branch points with no apparent dependence on proteasome inhibition. Our results demonstrate an effective strategy for detecting and characterizing the dynamics of branched conjugates under different cellular conditions.
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DOI：
10.1007/s13361-013-0789-4
发表时间：
2014-03
影响因子：
3.2
作者：
Guner, Huseyin;Close, Patrick L.;Cai, Wenxuan;Zhang, Han;Peng, Ying;Gregorich, Zachery R.;Ge, Ying
通讯作者：
Ge, Ying
影响因子：
64.5
作者：
Koegl, M;Hoppe, T;Jentsch, S
通讯作者：
Jentsch, S
影响因子：
4.8
作者：
Jacobson, Andrew D.;Zhang, Nan-Yan;Liu, Chang-Wei
通讯作者：
Liu, Chang-Wei
影响因子：
11.4
作者：
Kim, Hyoung Tae;Kim, Kwang Pyo;Goldberg, Alfred L.
通讯作者：
Goldberg, Alfred L.
影响因子：
64.8
作者：
Ikeda, Fumiyo;Deribe, Yonathan Lissanu;Skanland, Sigrid S.;Stieglitz, Benjamin;Grabbe, Caroline;Franz-Wachtel, Mirita;van Wijk, Sjoerd J. L.;Goswami, Panchali;Nagy, Vanja;Terzic, Janos;Tokunaga, Fuminori;Androulidaki, Ariadne;Nakagawa, Tomoko;Pasparakis, Manolis;Iwai, Kazuhiro;Sundberg, John P.;Schaefer, Liliana;Rittinger, Katrin;Macek, Boris;Dikic, Ivan
通讯作者：
Dikic, Ivan