Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin
Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin
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DOI：
10.1002/pmic.200401089
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发表时间：
2005-05-01
期刊：
影响因子：
3.4
通讯作者：
Gandolfi, AJ
中科院分区：
文献类型：
--
作者：
Kirkpatrick, DS;Weldon, SF;Gandolfi, AJ
建立了一种从人细胞中纯化和鉴定泛素(Ub)修饰的特异性蛋白质的蛋白质组学方法。在纯化的样品中，利用SEQUEST(R)的LC-MS/MS光谱鉴定了Ub和其他21个蛋白质，这些蛋白质包括Ub的几个预期载体，包括Ub结合酶和组蛋白蛋白。为了进行这些实验，通过稳定地转染HEK293细胞，获得了标记His(6X)-Ub和绿色荧光蛋白(GFP)的共表达表位的细胞系。泛素化蛋白经镍亲和层析纯化后，用胰酶消化。采用反相色谱分离复杂多肽混合物，采用LC-MS/MS纳米级联用技术，采用LC-Q四极离子陷阱质谱仪进行分析。将从His(6X)-Ub-G F P转基因细胞中鉴定的蛋白质与HEK293细胞中的一系列蛋白质进行比较，这些蛋白质在没有His标记的Ub的情况下与NiNTA琼脂糖凝胶结合。在原理验证实验中，用AS(111)(10LAM，24小时)处理His(6X)-Ub-GFP转基因细胞，以鉴定被Ub修饰的底物。在这个实验中，增殖细胞核抗原，一种DNA修复蛋白和已知的泛素底物，被自信地鉴定出来。这种蛋白质组学方法是为分析泛素化蛋白质而开发的，是朝着大规模表征多种生理和病理状态下的Ub-蛋白质结合物迈出的一步。
A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST (R) These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His (6X)-Ub-G F P transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (111) (10 lam, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.