Tagwerker Christian, Flick Karin, Cui Meng, Guerrero Cortnie, Dou Yimeng, Auer Bernhard, Baldi Pierre, Huang Lan, Kaiser Peter
Department of Biological Chemistry, School of Medicine, University of California Irvine, California 92697-1700, USA.
Mol Cell Proteomics. 2006 Apr;5(4):737-48. doi: 10.1074/mcp.M500368-MCP200. Epub 2006 Jan 23.
Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for two-step purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 m urea, by Ni(2+) chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein complexes, thereby expanding proteomic analyses to the description of weakly or transiently associated protein complexes.
串联亲和策略可实现卓越的蛋白质纯化水平，并显著改善了基于质谱的蛋白质组学实验结果。然而，目前的串联亲和标签在完全变性条件下与两步纯化不兼容。这种严格的纯化条件对于蛋白质修饰的质谱分析是理想的，因为它们能最大程度地保留翻译后修饰。在此，我们描述了组氨酸 - 生物素（HB）标签，这是一种用于在变性条件下进行两步纯化的新型串联亲和标签。HB标签由一个六组氨酸标签和一个细菌来源的体内生物素化信号肽组成，该信号肽可在酵母和哺乳动物细胞中诱导生物素高效附着于HB标签。带有HB标签的蛋白质可以在完全变性条件下，如8M尿素中，通过镍离子螯合色谱法和与链霉亲和素树脂结合进行顺序纯化。与HB标签兼容的严格分离条件可防止蛋白质修饰的丢失，并且串联亲和策略实现的高纯化水平有助于对翻译后修饰进行质谱分析。泛素化是一种特别敏感的蛋白质修饰，在天然条件下纯化过程中由于泛素水解酶活性会迅速丢失。HB标签是研究泛素化的理想选择，因为变性条件会抑制水解酶活性，并且串联亲和策略可大大降低非特异性背景。我们在酵母的全蛋白质组泛素谱分析实验中测试了HB标签，并鉴定出了一些已知的泛素化蛋白质以及迄今为止未鉴定的候选泛素化靶点。此外，与HB标签兼容的严格纯化条件允许对体内交联的蛋白质复合物进行有效的质谱鉴定，从而将蛋白质组学分析扩展到对弱相关或瞬时相关蛋白质复合物的描述。
