Yanagida M, Miura Y, Yagasaki K, Taoka M, Isobe T, Takahashi N
Applied Biological Science, Tokyo University of Agriculture and Technology, Japan.
Electrophoresis. 2000 May;21(9):1890-8. doi: 10.1002/(SICI)1522-2683(20000501)21:9<1890::AID-ELPS1890>3.0.CO;2-7.
We describe efficient methods for using functional proteomics analysis to study signal transduction pathways in murine fibroblast L929 cells following stimulation with tumor necrosis factor (TNF)-alpha. After stimulation with TNF-alpha, cellular proteins of L929 cells were extracted with a lysis buffer containing 0.3% sodium dodecyl sulfate (SDS) for 10-30 min time intervals, and were separated by two-dimensional (2-D) electrophoresis followed by immunoblot analysis with anti-phosphotyrosine antibody and alkaline phosphatase-anti IgG antibody conjugate. To improve detection sensitivity by immunoblot analysis we used a chemifluorescent substrate for alkaline phosphatase. One hundred protein spots were detected in the TNF-alpha stimulated L929 cell extract by immunoblot analysis. The use of chemifluorescence allowed us to quantitate immunoblotted spots with fluoroscanner so that we were able to detect time-dependent changes of a number of immunoblotted spots. Protein spots on a silver-stained 2-D gel corresponding to those detected by immunoblot analysis were subjected to in-gel trypsin digestion- matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis, respectively. Twenty-one proteins detected by immunoblot analysis were identified by MS-Fit database search analysis. Among them, the proteins that show time-dependent changes in staining intensity include vimentin, tubulin beta-chain, eukaryotic translation initiation factor 1A, chromatin assembly factor 1 (P48 subunit), probable protein disulfide isomerase P5, and several other proteins. Vimentin and tubulin beta-chain have been reported to be phosphorylated at tyrosine residues and involved in the signal transduction pathway induced by TNF-alpha. However, the other proteins have no previously known function in the signal transduction pathway. Thus, the methods used in this study seem to be suitable for the identification of time-dependent changes in many proteins that are involved in signal transduction. Usefulness of the method for comprehensive analysis of the proteins involved in signal transduction pathway and the limitations of the method are discussed.
我们描述了利用功能蛋白质组学分析来研究肿瘤坏死因子（TNF）-α刺激后小鼠成纤维细胞L929细胞中信号转导途径的有效方法。用TNF-α刺激后，在含有0.3%十二烷基硫酸钠（SDS）的裂解缓冲液中以10 - 30分钟的时间间隔提取L929细胞的细胞蛋白，然后通过二维（2-D）电泳分离，接着用抗磷酸酪氨酸抗体和碱性磷酸酶-抗IgG抗体偶联物进行免疫印迹分析。为了提高免疫印迹分析的检测灵敏度，我们使用了碱性磷酸酶的化学荧光底物。通过免疫印迹分析在TNF-α刺激的L929细胞提取物中检测到100个蛋白点。化学荧光的使用使我们能够用荧光扫描仪对免疫印迹的斑点进行定量，从而能够检测许多免疫印迹斑点的时间依赖性变化。对应于免疫印迹分析检测到的那些斑点，对银染2-D凝胶上的蛋白点分别进行胶内胰蛋白酶消化 - 基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱分析。通过MS-Fit数据库搜索分析鉴定了通过免疫印迹分析检测到的21种蛋白质。其中，染色强度呈现时间依赖性变化的蛋白质包括波形蛋白、微管蛋白β链、真核翻译起始因子1A、染色质组装因子1（P48亚基）、可能的蛋白质二硫键异构酶P5以及其他几种蛋白质。据报道，波形蛋白和微管蛋白β链在酪氨酸残基处被磷酸化，并参与TNF-α诱导的信号转导途径。然而，其他蛋白质在信号转导途径中以前没有已知的功能。因此，本研究中使用的方法似乎适用于鉴定参与信号转导的许多蛋白质的时间依赖性变化。讨论了该方法在信号转导途径中涉及的蛋白质综合分析中的实用性以及该方法的局限性。
