Mass spectrometric characterization of the affinity-purified human 26S proteasome complex
Mass spectrometric characterization of the affinity-purified human 26S proteasome complex
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DOI：
10.1021/bi061994u
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发表时间：
2007-03-20
期刊：
影响因子：
2.9
通讯作者：
Huang, Lan
中科院分区：
文献类型：
--
作者：
Wang, Xiaorong;Chen, Chi-Fen;Huang, Lan
26S蛋白酶体是一种多亚基复合物，负责降解泛素化底物，在调节各种生物过程中发挥着关键作用。为了充分了解蛋白酶体复合物的功能和调节，重要的一步是阐明其亚基组成和翻译后修饰。为了实现这一目标，我们开发了一种新的亲和纯化策略，使用 HB 标签的衍生物来快速分离人 26S 蛋白酶体复合物，以进行后续的蛋白质组分析。该复合物的纯化是通过表达 HB 标记蛋白酶体亚基的稳定 293 细胞系并通过高亲和力链霉亲和素结合 TEV 裂解洗脱来实现的。通过 LC-MS/MS 鉴定了 26S 蛋白酶体复合物的完整组成，包括最近指定的新亚基。此外，还鉴定了所有已知的蛋白酶体激活蛋白和参与泛素-蛋白酶体降解途径的成分。除了亚基组成外，蛋白酶体亚基的 N 端修饰和磷酸化也已得到表征。已鉴定出来自 8 个亚基的 12 个新磷酸化位点，并确定了 25 个亚基的 N 末端修饰，其中 12 个亚基以前未在哺乳动物中报道过。我们还观察到 Rpn2 亚基的不同 N 末端加工，从而识别出该蛋白质的两个不同 N 末端。这项工作首次通过亲和纯化和串联质谱法全面表征了人类 26S 蛋白酶体复合物。这里获得的详细蛋白质组分析对于未来旨在全面了解人类 26S 蛋白酶体复合物的结构-功能关系的研究具有重要意义。
The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.