Guy G R, Philip R, Tan Y H
Signal Transduction Laboratory, National University of Singapore.
Electrophoresis. 1994 Mar-Apr;15(3-4):417-40. doi: 10.1002/elps.1150150160.
Two-dimensional (2-D) gel electrophoresis has been used to map proteins from various cell types in an effort to eventually link such maps to the sequencing of the entire human genome. While this analysis indicates the cellular disposition and expression of proteins, another application of 2-D gels, the analysis of phosphoproteins, can provide much information as to the assembly and "wiring" of the signal transduction circuits within cells which appear to be enervated by phosphate exchange. The preparation and separation of 32P-labeled proteins is described, as well as various analytical methods, including: the variety of gel systems available for specialist types of analyses, comparing 33P- and 32P-labeling of proteins, imaging techniques, phosphoamino analysis, phosphopeptide separation, identifying the amino acid groups that are phosphorylated, and the identification of phosphoproteins on 2-D gels by immunoprecipitation, corunning of purified proteins, comparative mapping and microsequencing, and by Western blotting. Examples (in brackets) are given of applications in which 2-D phosphogels can be applied, which offer advantages over other techniques. These include: (i) identifying in vivo substrates for kinases (protein kinase C activated by phorbol myristate acetate), (ii) investigating cytokine signaling pathways (tumor necrosis factor and interleukin-1), (iii) investigating the effects of drugs on signaling pathways (okadaic acid, menadione and cyclooxygenase inhibitors), (iv) characterization of specific phosphoproteins (heat-shock protein Hsp27 and stathmin), (v) comparing normal and transformed cells (MRC-5 human lung fibroblasts and their SV-40-transformed counterparts, MRC-5 SV1 cells), (vi) purifying phosphoproteins, (vii) investigating the relationship of protein phosphorylation to stages in the cell cycle (stathmin), (viii) investigating protein/protein interactions, (ix) mapping in vitro kinase substrates (protein kinase C, protein kinase A, and mitogen activated protein kinase activated protein kinase 2), and (x) locating and identifying cellular phosphatases (Hsp27 phosphatase). It is possible that the mapping of phosphoproteins can be linked to other 2-D gel databases and that information derived from these can be used in the future to better understand the signaling mechanisms of normal and cancerous cells.
二维（2-D）凝胶电泳已被用于绘制各种细胞类型的蛋白质图谱，以期最终将这些图谱与整个人类基因组的测序联系起来。虽然这种分析揭示了蛋白质的细胞定位和表达，但二维凝胶的另一项应用，即磷蛋白分析，能够提供大量有关细胞内信号转导回路的组装和“布线”的信息，这些信号转导回路似乎通过磷酸盐交换而被激活。本文描述了32P标记蛋白质的制备和分离方法，以及各种分析方法，包括：可用于特定类型分析的多种凝胶系统、比较蛋白质的33P和32P标记、成像技术、磷氨基酸分析、磷酸肽分离、鉴定被磷酸化的氨基酸基团，以及通过免疫沉淀、纯化蛋白质的共电泳、比较图谱和微量测序，以及蛋白质印迹法在二维凝胶上鉴定磷蛋白。文中给出了（括号内）二维磷酸凝胶可应用的示例，这些应用相较于其他技术具有优势。其中包括：（i）鉴定激酶的体内底物（佛波酯肉豆蔻酸酯激活的蛋白激酶C），（ii）研究细胞因子信号通路（肿瘤坏死因子和白细胞介素-1），（iii）研究药物对信号通路的影响（冈田酸、甲萘醌和环氧化酶抑制剂），（iv）特定磷蛋白的表征（热休克蛋白Hsp27和微管相关蛋白），（v）比较正常细胞和转化细胞（MRC-5人肺成纤维细胞及其SV-40转化的对应细胞，MRC-5 SV1细胞），（vi）纯化磷蛋白，（vii）研究蛋白质磷酸化与细胞周期各阶段的关系（微管相关蛋白），（viii）研究蛋白质/蛋白质相互作用，（ix）绘制体外激酶底物图谱（蛋白激酶C、蛋白激酶A和丝裂原活化蛋白激酶激活的蛋白激酶2），以及（x）定位和鉴定细胞磷酸酶（Hsp27磷酸酶）。磷蛋白图谱有可能与其他二维凝胶数据库相联系，并且未来可以利用从这些数据库中获得的数据更好地理解正常细胞和癌细胞的信号传导机制。
