Developing a Bimolecular Affinity Purification Strategy to Isolate 26S Proteasome Holocomplexes for Complex-Centric Proteomic Analysis.
Developing a Bimolecular Affinity Purification Strategy to Isolate 26S Proteasome Holocomplexes for Complex-Centric Proteomic Analysis.
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开发一种双分子亲和力纯化策略，以分离26S蛋白酶体的全圆形复合体，以进行复杂的蛋白质组学分析。
DOI：
10.1021/acs.analchem.1c03551
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发表时间：
2021-10-05
影响因子：
7.4
通讯作者：
Huang L
中科院分区：
文献类型：
--
作者：
Yu C;Wang X;Li W;Liu Y;Huang L
26S蛋白酶体是一种巨道尔顿蛋白复合物，负责真核生物中大部分的细胞内降解。它由两个亚复合物组成：20 S核心颗粒和19 S调节颗粒，它们在细胞中形成组成和结构异质的蛋白酶体复合物。为了充分表征26S蛋白酶体，有必要了解其结构和功能的差异。近年来，多种质谱方法已被开发用于蛋白酶体结构动力学的研究，其中生物化学分离的复合物进行分析。由于蛋白酶体复合物的固有异质性，单诱饵亲和纯化通常导致组成异质复合物的混合物，而不管诱饵如何，使得复合物特异性构象和功能的准确评估具有挑战性。为了促进复合物为中心的分析，我们采用了双分子亲和纯化方法，利用双诱饵细胞系表达标记的19S和标记的20S亚基，以提高所得的26S holocomplexes的均匀性。为了建立该方法，进行了四种类型的纯化，并通过生化分析和两种无标记定量MS方法对所得样品进行了广泛的检查。我们的研究结果表明，这种纯化策略的有效性，以提高复杂的同质性，下游生化和MS表征。这种策略将有助于在不同条件下对复合物特异性分子细节进行详细的定量评估，并可直接用于研究其他复合物。
The 26S proteasome is a mega-dalton protein complex responsible for the majority of intracellular degradation in eukaryotes. It is composed of two subcomplexes: the 20S core particle and 19S regulatory particle, which form compositionally and structurally heterogeneous proteasome complexes in cells. To fully characterize the 26S proteasome, it is necessary to understand its structural and functional diversities. Multiple mass spectrometric methodologies have been developed in recent years for the study of proteasome structural dynamics, in which biochemically isolated complexes are subjected for analysis. Due to the inherent heterogeneity of proteasome complexes, single-bait affinity purification typically results in a mixture of compositionally heterogeneous complexes regardless of the baits, making accurate assessment of complex-specific conformations and functions challenging. To facilitate complex-centric analysis, we have adopted a bimolecular affinity purification method utilizing a dual-bait cell line expressing a tagged 19S and a tagged 20S subunit to improve the homogeneity of the resulting 26S holocomplexes. To establish the method, four types of purifications were performed and the resulting samples were extensively examined by biochemical analysis and two label-free quantitative MS methods. Our results have demonstrated the effectiveness of this purification strategy to improve complex homogeneity for downstream biochemical and MS characterizations. This strategy will be valuable for facilitating detailed quantitative assessments of complex-specific molecular details under different conditions and can be directly adopted for studying other complexes.
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影响因子：
9.9
作者：
Snider J;Kotlyar M;Saraon P;Yao Z;Jurisica I;Stagljar I
通讯作者：
Stagljar I
影响因子：
7.3
作者：
Wang X;Yen J;Kaiser P;Huang L
通讯作者：
Huang L
DOI：
10.1074/mcp.r110.003871
发表时间：
2011-05-01
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Lee, Min Jae;Lee, Byung-Hoon;Finley, Daniel
通讯作者：
Finley, Daniel
影响因子：
4.8
作者：
Wang, Xiaorong;Chemmama, Ilan E.;Huang, Lan
通讯作者：
Huang, Lan
蛋白酶体：结构和功能的概述。
DOI：
10.2183/pjab.85.12
发表时间：
2009
期刊：
Proceedings of the Japan Academy. Series B, Physical and biological sciences
影响因子：
--
作者：
Tanaka K
通讯作者：
Tanaka K